Anti RFP

Otoferlin is phosphorylated by CaMKIIδ in vitro. (A) Otoferlin fragments C2ABC (aa 1616 in NP_001093865, 70 kDa) and C2DEF (aa 9081932, 118 kDa), were expressed in E. coli and subjected to an in vitro phosphorylation assay with CaMKIIδ and Ca2+/calmodulin. Reactions were stopped after 5 min of incubation and proteins were run on a Coomassie gel. Note the slight shift in mass of the fragments between experiment (lane 2) and control without kinase (lane 3). Coomassie stained bands corresponding to otoferlin C2DEF and C2ABC were cut off the gel and processed for mass spectrometric analysis of otoferlin phosphorylation (Supplementary Figure S2). (B) Three independent experiments as in (A) revealed 10 serine/threonines in otoferlin that were reproducibly phosphorylated by CaMKIIδ. The putative otoferlin domain topology (in mouse isoform 1; NP_001093865) predicts six C2 domains (C2A to C2F; purple), a coiled coiled domain (orange), a FerB domain (yellow), and a transmembrane domain (TM) (dark gray). Five of the phosphorylation sites are located in C2 domains. Figure provided by CiteAb. Source: Front Synaptic Neurosci, PMID: 29046633.

Immunoprecipitation and western blot show interaction of otoferlin with CaMKIIδ. (AC) Two HA-tagged mouse otoferlin fragments, C2ABC (aa 1632 in NP_001093865; 70 kDa) and C2DEF (aa 9331920; 114 kDa) were co-transfected with mcherry-tagged mouse CaMKIIδ into HEK293 cells. Transfections were performed either with otoferlin C2ABC and CaMKIIδ (A, Input Lane 1 and 2), otoferlin C2DEF and CaMKIIδ (B, Input Lane 1 and 2) or in the presence of both C2ABC and C2DEF fragments and CaMKIIδ (C, Input Lane 1 and 2). Co-immunoprecipitations of C2ABC-HA and C2DEF-HA were conducted from HEK293 cell lysates using anti-HA antibodies. CaMKIIδ-mcherry was detected in the eluate using an anti-RFP (red fluorescent protein) antibody (AC, Lane 3), indicating that CaMKIIδ co-precipitated with recombinant otoferlin fragments. Figure provided by CiteAb. Source: Front Synaptic Neurosci, PMID: 29046633.

Immunoprecipitation and western blot show interaction of otoferlin with CaMKIIδ. (AC) Two HA-tagged mouse otoferlin fragments, C2ABC (aa 1632 in NP_001093865; 70 kDa) and C2DEF (aa 9331920; 114 kDa) were co-transfected with mcherry-tagged mouse CaMKIIδ into HEK293 cells. Transfections were performed either with otoferlin C2ABC and CaMKIIδ (A, Input Lane 1 and 2), otoferlin C2DEF and CaMKIIδ (B, Input Lane 1 and 2) or in the presence of both C2ABC and C2DEF fragments and CaMKIIδ (C, Input Lane 1 and 2). Co-immunoprecipitations of C2ABC-HA and C2DEF-HA were conducted from HEK293 cell lysates using anti-HA antibodies. CaMKIIδ-mcherry was detected in the eluate using an anti-RFP (red fluorescent protein) antibody (AC, Lane 3), indicating that CaMKIIδ co-precipitated with recombinant otoferlin fragments. Figure provided by CiteAb. Source: Front Synaptic Neurosci, PMID: 29046633.

Transgenic model and effects of acute 4D overexpression on NSC and progenitors of the SVZDrawings of the nestinCreERT2 (Imayoshi et？al, 2008), ROSA26rtTA‐flox (Belteki et？al, 2005), and tet4D‐RFP (Nonaka‐Kinoshita et？al, 2013) alleles of the 4D line.From top to bottom: experimental design of 4D induction, fluorescence pictures of the SVZ of a 4D+ mouse and quantification of the proportion of RFP− (black) and RFP+ (red) progenitors among B1, C, and A cells identified with markers as indicated.From top to bottom: experimental design, fluorescence pictures of the SVZ of a 4D− (left) and 4D+ (right and insets magnified) mice, and quantification of the proportion of BrdU+ among B1, C, and A cells (identified as in B). Note that in 4D+ mice quantification was restricted to the RFP+ subpopulation (red bars).Quantification of the absolute number of B1, C, and A cells (identified as in B) in the SVZ of 4D− (white) and 4D+ (black) mice regardless of RFP expression (RFP+/−).Data information: (BD) Mean？±？SEM; *P？<？0.05 and **P？<？0.01 calculated by unpaired Student's t‐test; N？=？3 mice, n？>？423 cells for each quantification. Scale bar？=？50？μm (B and C) or 20？μm (inset in C). Figure provided by CiteAb. Source: EMBO J, PMID: 30643018.

Transgenic model and effects of acute 4D overexpression on NSC and progenitors of the SVZDrawings of the nestinCreERT2 (Imayoshi et？al, 2008), ROSA26rtTA‐flox (Belteki et？al, 2005), and tet4D‐RFP (Nonaka‐Kinoshita et？al, 2013) alleles of the 4D line.From top to bottom: experimental design of 4D induction, fluorescence pictures of the SVZ of a 4D+ mouse and quantification of the proportion of RFP− (black) and RFP+ (red) progenitors among B1, C, and A cells identified with markers as indicated.From top to bottom: experimental design, fluorescence pictures of the SVZ of a 4D− (left) and 4D+ (right and insets magnified) mice, and quantification of the proportion of BrdU+ among B1, C, and A cells (identified as in B). Note that in 4D+ mice quantification was restricted to the RFP+ subpopulation (red bars).Quantification of the absolute number of B1, C, and A cells (identified as in B) in the SVZ of 4D− (white) and 4D+ (black) mice regardless of RFP expression (RFP+/−).Data information: (BD) Mean？±？SEM; *P？<？0.05 and **P？<？0.01 calculated by unpaired Student's t‐test; N？=？3 mice, n？>？423 cells for each quantification. Scale bar？=？50？μm (B and C) or 20？μm (inset in C). Figure provided by CiteAb. Source: EMBO J, PMID: 30643018.

Chronic effect of 4D overexpression on NSC and OB neurogenesisAExperimental design used to assess the chronic effect of a transient 4D induction with BrdU and EdU given during Dox administration or 1？h before sacrifice, respectively.BEFrom top to bottom: fluorescence pictures of the SVZ (BD) or OB (E) and absolute number (B, C, and E) or proportions (CE) of cells in 4D− (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. Insets in (C) are magnified (right) with arrowheads pointing label‐retaining NSC (white) or astrocytes (empty). Arrowheads in (D) point cell doublets (among RFP+ protein‐retaining cells in 4D+). (E) GL, glomerular; EPL, external plexiform; MCL, mitral cell and GCL, granule cell layers.Data information: (BE) Mean？±？SEM; *P？<？0.05, **P？<？0.01 assessed by unpaired Student's t‐test (bar graphs) or Fisher's exact test (pie graphs); N？=？3 mice, n？>？285 cells for each quantification. Scale bars？=？50？μm (B, C, and E) and 20？μm (D and insets in C). Figure provided by CiteAb. Source: EMBO J, PMID: 30643018.

Characterization of the transgenic model and effect of 4D on the RMSA Fluorescence image of a sagittal section of a 4D+ brain after a 4‐day treatment with doxycycline showing RFP signal confined to the SVZ and RMS (nuclei counterstained with DAPI; blue). Insets show representative images of specific brain regions (as indicated) and the olfactory epithelium. APhase contrast picture of the SVZ upon in？situ hybridization against mRNA for RFP in a 4D+ brain treated as in (A) and sacrificed immediately after (left) or 2？days after (right) doxycycline administration. (B, C) Experimental design (top), fluorescence pictures (left with magnified insets), and quantifications (right) of BrdU incorporation in the RMS (B) or SVZ (C). (B) shows the proportion of BrdU in C (Mash1+) and A (DCX+) cells in 4D− (white) and 4D+ (red; among RFP+) mice. (C) shows the proportion of RFP− (black) and RFP+ (red) among BrdU+ cells of 4D+ mice. (A) OB, olfactory bulb; RMS, rostral migratory stream; LV, lateral ventricle; DG, dentate gyrus; OE, olfactory epithelium. (AC) Tam, tamoxifen; Dox, doxycycline. (B, C) Mean？±？SEM; **P？<？0.01; unpaired Student's t‐test; N？=？3 mice and n？>？1,100 cells. Scale bars？=？500 (A top, A), 100 (insets A and A), 50 (B and C), and 20 (insets B and C) μm. Figure provided by CiteAb. Source: EMBO J, PMID: 30643018.

Characterization of the transgenic model and effect of 4D on the RMSA Fluorescence image of a sagittal section of a 4D+ brain after a 4‐day treatment with doxycycline showing RFP signal confined to the SVZ and RMS (nuclei counterstained with DAPI; blue). Insets show representative images of specific brain regions (as indicated) and the olfactory epithelium. APhase contrast picture of the SVZ upon in？situ hybridization against mRNA for RFP in a 4D+ brain treated as in (A) and sacrificed immediately after (left) or 2？days after (right) doxycycline administration. (B, C) Experimental design (top), fluorescence pictures (left with magnified insets), and quantifications (right) of BrdU incorporation in the RMS (B) or SVZ (C). (B) shows the proportion of BrdU in C (Mash1+) and A (DCX+) cells in 4D− (white) and 4D+ (red; among RFP+) mice. (C) shows the proportion of RFP− (black) and RFP+ (red) among BrdU+ cells of 4D+ mice. (A) OB, olfactory bulb; RMS, rostral migratory stream; LV, lateral ventricle; DG, dentate gyrus; OE, olfactory epithelium. (AC) Tam, tamoxifen; Dox, doxycycline. (B, C) Mean？±？SEM; **P？<？0.01; unpaired Student's t‐test; N？=？3 mice and n？>？1,100 cells. Scale bars？=？500 (A top, A), 100 (insets A and A), 50 (B and C), and 20 (insets B and C) μm. Figure provided by CiteAb. Source: EMBO J, PMID: 30643018.

Characterization of the transgenic model and effect of 4D on the RMSA Fluorescence image of a sagittal section of a 4D+ brain after a 4‐day treatment with doxycycline showing RFP signal confined to the SVZ and RMS (nuclei counterstained with DAPI; blue). Insets show representative images of specific brain regions (as indicated) and the olfactory epithelium. APhase contrast picture of the SVZ upon in？situ hybridization against mRNA for RFP in a 4D+ brain treated as in (A) and sacrificed immediately after (left) or 2？days after (right) doxycycline administration. (B, C) Experimental design (top), fluorescence pictures (left with magnified insets), and quantifications (right) of BrdU incorporation in the RMS (B) or SVZ (C). (B) shows the proportion of BrdU in C (Mash1+) and A (DCX+) cells in 4D− (white) and 4D+ (red; among RFP+) mice. (C) shows the proportion of RFP− (black) and RFP+ (red) among BrdU+ cells of 4D+ mice. (A) OB, olfactory bulb; RMS, rostral migratory stream; LV, lateral ventricle; DG, dentate gyrus; OE, olfactory epithelium. (AC) Tam, tamoxifen; Dox, doxycycline. (B, C) Mean？±？SEM; **P？<？0.01; unpaired Student's t‐test; N？=？3 mice and n？>？1,100 cells. Scale bars？=？500 (A top, A), 100 (insets A and A), 50 (B and C), and 20 (insets B and C) μm. Figure provided by CiteAb. Source: EMBO J, PMID: 30643018.

ELISA results of purified Mouse anti-RFP Monoclonal Antibody tested against RFP (p/n 000-001-379). Each well was coated in duplicate with 1.0 ug of the antigen. The starting dilution of antibody was 5μg/ml and the X-axis represents the Log10 of a 3-fold dilution. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using 3% fish gel, anti-Mouse IgG Antibody Peroxidase Conjugated Secondary and TMB ELISA Peroxidase Substrate (p/n TMBE-1000).