proteintech 13573 1 ap rabbit anti ccdc22  (Proteintech)

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: (A) Urinary β2-microglobulin level of individuals with biallelic pathogenic mutations in VPS35L or CCDC93 . (B) The schematic illustration depicts the chimeric constructs of Human-LRP2 utilized in the study. In addition to the wild-type cytoplasmic sequence, mutant constructs were generated in which either or both NPxY motifs were substituted with NPxA. (C) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of LRP2. GFP-tagged cytoplasmic tail of CI-MPR, a SNX-BAR cargo protein, was used as negative control. Bar graphs show band intensities relative to WT calculated from three independent experiments. (D) Representative blots for LRP1, LRP2, and N-Cadherin from three independent experiments. Cell surface protein fractions were obtained from HEK293T cell lines. Bar graphs show relative values of KO cells to their rescue or parental cells. (E) Representative view of mCherry-D3 uptake in parental, VPS35L-KO, and VPS35L-rescue cells. Cells were incubated with mCherry-D3 for 30 min, followed by DAPI staining and imaging with a fluorescence microscope. mCherry intensity was quantified using Image J software. 10 fields were acquired in each condition in each of three independent experiments, and mCherry intensity of each field was normalized to the number of DAPI-stained nuclei. Scale bars, 10 µm. (F) HEK293T cell lines were incubated with mCherry-D3 for 30 min followed by FACS analysis to quantitate cellular uptake of mCherry-D3. (G) Immunohistochemistry of Lrp2 in renal tissue in littermate control or Vps35l-cKO Nestin . Red arrows indicate Lrp2 in S1 segment of proximal tubules. Three mice were analyzed in each group. G; Glomerulus. (H) Schematic illustration of the molecular mechanism underlying the proteinuria observed in Ritscher-Schinzel syndrome. (C, D, E) Error bars represent mean± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Construct, Sequencing, Mutagenesis, Generated, Over Expression, Negative Control, Incubation, Staining, Imaging, Fluorescence, Microscopy, Software, Immunohistochemistry, Control

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: Loss of membrane proteins such as LRP4 and FGFR2 involved in disease pathogenesis in skeletal system in Ritscher-Schinzel syndrome. (A) Representative blots of Co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of Human-LRP4 from three independent experiments. The interaction between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of LRP4 was significantly decreased when NPxY was substituted with NPxA. Bar graphs show relative density of the bands in immunoprecipitated proteins from three independent experiments. (B) Representative blots of LRP4 in cell surface protein fraction obtained from H4 cell line. N-Cadherin was used for loading control. Bar graph shows relative intensity of knock-out cells to their rescue or parental cells among three independent experiments. (C) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in TMT-based proteomics in sh-SNX17 comparing to sh-SCR in MC3T3-E1 mouse osteoblast cells from three independent experiments. Two independent sh-RNAs for SNX17 were used to avoid off-target effects. (D) Representative blots for FLAG-nanotrap of FLAG-SNX17 under co-overexpression of chimeric constructs of FGFR2 cytoplasmic tail from three independent experiments. NxxY motifs were mutated to NxxA in the mutant. (E) Representative blots for FGFR2 in MC3T3-E1. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of sh-SNX17 or sh-VPS35L knock-down cells compared to sh-SCR control cell (n=3). (F) Representative blots for ERK and pERK under stimulation of FGF2 in MC3T3-E1 from three independent experiments. Cells were cultured with 5ng/ml of FGF-2 overnight, then media was replaced with fresh media with 100ng/ml of FGF-2 for 7 min. Cells were lysed and analysed by western blotting. (G) Representative pictures and scatter plots of their body weight in VPS35L Prx1 -cKO mice and their littermate controls. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (H) Representative images and graph showing length of tibiae in mice at 8 weeks with indicated genotype. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (I) Gene enrichment analysis of downregulated genes in Vps35l-cKO Prx1 compared to their littermate controls was performed using RNA sequencing analysis data. Total RNA was extracted from E16.5 mice chondrocytes. [Control; n=5, Vps35l-cKO Prx1 ; n=3]. (J) Gene enrichment analysis of gene sets, where genes upregulated by FGF2 stimulation in littermate controls but not in Vps35l-cKO Prx1 were included. Tibiae obtained from E16.5 mouse embryos of either Vps35l-cKO Prx1 or their littermate controls were cultured for four days with or without FGF2, followed by RNA extraction for RNA sequencing analysis. [Control; n=3, Vps35l-cKO Prx1 ; n=4]. (A, B, D, E, F) Error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Membrane, Expressing, Immunoprecipitation, Control, Knock-Out, Over Expression, Construct, Mutagenesis, Knockdown, Cell Culture, Western Blot, RNA Sequencing Assay, RNA Extraction

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: SNX17-Retriever/CCC/WASH pathway is essential for recycling of Reelin signaling receptors, APP family, and SLITRK family proteins. (A) Representative blots of co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of LRP8 and VLDLR from three independent experiments. The interactions between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of Human-LRP8 and Human-VLDLR were significantly decreased when NPxY was substituted with NPxA. (B) Representative blots of analysis from three independent experiments using DIV17 rat cortical neurons transduced with either a scramble-control, SNX17, or VPS35L shRNA. Bar graphs show relative protein abundance of LRP8 and VLDLR in cell lysate or cell surface. (C) DIV17 rat cortical neurons transduced with shRNA were incubated for 30 min with or without AP-Reelin, followed by cell lysis and western blot analysis. Bar graph shows quantification of band intensities of AP relative to cells transduced with sh-SCR control from three independent experiments. (D) DIV17 rat cortical neurons transduced with shRNA were incubated for 7 min with or without Reelin. Phosphorylation level of Dab1 was then measured using immunoprecipitation and western blot analysis. Representative blots and quantification from three independent analyses are shown. (E) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in sh-VPS35L suppression comparedto sh-SCR in DIV17 rat cortical neuron from three independent TMT-based proteomic experiments. Two independent sh-RNAs for VPS35L were used to avoid off-target effects. (F) Enrichment analysis of significantly downregulated proteins (LogFC < −0.32, p < 0.05) in the sh-VPS35L compared to sh-SCR by Metascape. (G) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-SLITRK family proteins from three independent experiments. All proteins except for SLITRK4 have an NPxY motif, which was mutated to NPxA in the mutant. (H) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-APP family proteins from three independent experiments. APP, APLP1, and APLP2 have NPxY motifs, and the NPxY motif was mutated to NPxA in the mutant. (I) Representative blots for APP and APLP2 in rat cortical neurons. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of cells with sh-SNX17 or sh-VPS35L suppression compared to sh-SCR control cells (n=3). In all graphs, error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Expressing, Transduction, Control, shRNA, Incubation, Lysis, Western Blot, Immunoprecipitation, Over Expression, Construct, Mutagenesis

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: Loss of Retriever/CCC/WASH function disrupts SNX17-cargo recycling (A and B) Representative blots of H4 parental and KO/rescue for VPS35L, CCDC22, WASHC5, and SNX17 using whole cell lysate. β-actin was used as loading control. Bar graphs show relative protein abundance (n=3). (C) Representative view of Immunofluorescence staining of endogenous COMMD1 and VPS35 in H4 cell lines. Graph shows quantification of Pearson’s correlation score from three independent experiments. Pearson’s coefficients for individual cells and means are presented by smaller and larger circles, respectively. Cell numbers analyzed across experiments were 99 parental, 104 SNX17-KO, 99 VPS35L-KO, 108 CCDC22-KO, 135 WASHC5-KO cells. (D) Representative blots of H4 parental and KO/rescue for VPS35L, CCDC22, WASHC5, and SNX17 using cell surface fraction. N-Cadherin was used as loading control. Bar graphs show relative protein abundance (n=3). (E) Representative blots of HEK293T parental, VPS35L-KO, COMMD4-KO, and COMMD4-L41R knock-in cells. N-Cadherin was used as loading control. Bar graphs show relative protein abundance (n=3). (F) Schematic illustration to show accumulation of responsible genes for 3C/Ritscher-Schinzel syndrome in SNX17-Retriever/CCC/WASH recycling pathway. (B-E) Error bars represent mean± SD. **, P<0.01; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Control, Immunofluorescence, Staining, Knock-In

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: Loss of SNX17-Retriever/CCC/WASH pathway is a molecular root cause of Ritscher-Schinzel syndrome. (A) Representative blots of H4 parental and KO/rescue for VPS35L, COMMD4, COMMD9, and CCDC93. N-Cadherin and β-actin were used as loading controls. Bar graphs show relative protein abundance (n=3). (B) Representative view of immunofluorescence staining of endogenous COMMD1 and VPS35 in H4 cell lines. Scale bars, 10 µm and 2 µm, respectively. Graph shows quantification of Pearson’s correlation score from three independent experiments. Pearson’s coefficients for individual cells and means are presented by smaller and larger circles, respectively. Cell numbers analyzed across experiments were 91 parental, 86 COMMD4-KO, 93 COMMD9-KO and 90 CCDC93-KO cells. (C) Representative blots of H4 parental and KO/rescue for VPS35L, VPS26C and WASHC4. N-Cadherin and β-actin were used as loading control. Bar graphs show relative protein abundance (n=3). (D) Representative view of immunofluorescence staining of endogenous COMMD1 and VPS35 in H4 cell lines. Graph shows quantification of Pearson’s correlation score from three independent experiments. Pearson’s coefficients for individual cells and means are presented by smaller and larger circles, respectively. Cell numbers analyzed across experiments were 87 parental, 94 VPS26C-KO and 88 WASHC4-KO. (E) Mutations in VPS35L and VPS26C belonging to Retriever complex and COMMD4, COMMD9, CCDC22 and CCDC93 belonging to CCC complex associated with Ritscher-Schinzel syndrome and intellectual disability mapped onto the structure of Commander complex. (A-D) Error bars represent mean± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Immunofluorescence, Staining, Control

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: Cell surface proteome analysis in HEK293T cell identified SEZ6L2 and SNX17-cargo proteins affected in VPS35L, CCDC22, and WASHC5 knock-out. (A) Volcano plots of proteins with decreased (blue circles) or increased (red circles) cell surface abundance in knock-out HEK293T cell lines of VPS35L, CCDC22, and WASHC5, from four independent TMT-based proteomics experiments. (B) Volcano plots of proteins with decreased (blue circles) or increased (red circles) cell surface abundance in HEK293T wild-type rescue compared to their knock-out of VPS35L, CCDC22, and WASHC5, from four independent TMT-based proteomics experiments.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Knock-Out