anti-tbc1d4  (Millipore)

Journal: Redox Biology

Article Title: Revisiting insulin-stimulated hydrogen peroxide dynamics reveals a cytosolic reductive shift in skeletal muscle

doi: 10.1016/j.redox.2025.103607

Figure Lengend Snippet: Antibody information.

Article Snippet: TBC1D4 Thr642 , Cell Signaling Technology , 1:1000, 3 % skim milk , 4288.

Techniques:

Journal: bioRxiv

Article Title: Mapping the endosomal proximity proteome reveals Retromer as a hub for RAB GTPase regulation

doi: 10.1101/2024.11.22.622898

Figure Lengend Snippet: (A, B) Retromer interactions with TBC GAP and DENND GEF proteins across the human proteome. VPS26A-VPS35 and VPS29-VPS35 assemblies or individual VPS29, VPS26A and VPS35 proteins were screened against all human proteins with TBC and DENN domains using AlphaFold2. The confidence of potential interactors was scored based on the sum of the interfacial PTM score (iPTM) averaged from three models, pDOCKQ and SPOC scores. In TBC domain GAPs, TBC1D5 and TBC1D13 shows high confidence. In DENN domain GEFs, DENND4A and DENND4C were confidently predicted to bind to VPS29/Retromer. Others found to possess a PL motif but show low confidence in association with VPS29 binding are TBC1D1, TBC1D4 and DENND11. (C) GFP based co-immunoprecipitation (co-IP) of GFP-TBC1D1, GFP-TBC1D4 or GFP-TBC1D13 after transient transfection in HEK293T cells. Co-expression of GFP-TBC1D1 with mCherry-TBC1D4 increased pull-down of Retromer complex subunits. (D, E) GFP based co-IP after transient transfection in HEK293T cells of wild-type (WT) and PL interacting-motive mutant versions of GFP-TBC1D1 (P87A) and mCherry-TBC1D4 (P15A). Quantitation and statistical analysis of relative band intensity for the indicated proteins normalized to GFP band intensity. n = 3 independent experiments. T-test analysis, data presented as mean values relative to WT and error bars represent SD. (F) Schematic summarizing the interactions of Retromer with the identified TBC GAPs and DENND GEFs and the similarity with binding to the FAM21 subunit of the WASH complex and the RidL protein from legionella. The intramolecular occlusion of the equivalent binding site in VPS29 when assembled in the Retriever complex prevents Retriever from binding to any of these proteins through these specific mechanisms.

Article Snippet: Primary antibodies: β-Actin (Sigma-Aldrich, A1978; 1:5000 WB, 1:1000 IF), GAPDH (Sigma-Aldrich, G9545; 1:5000 WB), GAPDH (Sigma-Aldrich, G8795; 1:5000 WB), EEA1 (Cell Signalling; 3288S; 1:200 IF), GFP (Roche; 11814460001; clones 7.1/13.1; 1:1000 WB), mCherry/RFP (Abcam, 167453; 1:1000 WB), FLAG-M2 (Sigma, F1804; 1:1000 WB, 1:200 IF), GLUT1 (Abcam; ab115730; 1:1000 WB; 1:400 IF), LAMP1 (Developmental Studies Hybridoma Bank; AB_2296838; clone H4A3; 1:400 IF), VPS29 (Santa Cruz; D-1; sc-398874; 1:500 WB), VPS35 (Abcam, ab97545; 1:1000 WB, 1:400 IF), VPS35 ( Antibodies.com , A83699; 1:400 IF), VPS26 (Abcam, ab23892; 1:1000 WB), VPS35L (Abcam, ab97889; 1:1000 WB), VPS26C (Sigma-Aldrich, ABN87; 1:1000 WB), VPS35L (Invitrogen, PA5-28553; 1:200 IF), SNX1 (BD Transduction Lab, 611482; 1:1000 WB, 1:200 IF), SNX1 (Proteintech, 10304-1-AP; 1:200 IF), SNX2 (BD Transduction Lab, 611308; 1:1000 WB), SNX5 (Abcam, ab180520; 1:1000 WB), SNX6 (Santa Cruz, sc-365965; 1:1000 WB), SNX17 (Proteintech, 10275-1-AP; 1:1000 WB), SNX17 (Sigma, HPA043867, 1:100 IF), SNX27 (Proteintech, 16329-1-AP; 1:1000 WB), SNX27 (Abcam, ab77799; 1:100 IF), HRS/HGS (Enzo Life Sciences, ALX-804-382-C050; 1:2000 WB, 1:100 IF), STAM (Proteintech, 12434-1-AP; 1:2000 WB, 1:100 IF), FAM21 (Gift from Dan Billadeau; 1:2000 WB), Integrin-α5 (Abcam, ab150361; 1:1000 WB, 1;200 IF), DENND4C (Sigma-Aldrich, HPA014917, 1:1000 WB, 1:500 IF), DENND4C (StressMarq Bioscience, SMC-610, 1:100 IF), DENND4A (Abcam, ab117758, 1:500 WB), HAUS2 (ThermoFisher, PA5-31258; 1:500 WB), HAUS6 (Proteintech, 16933-1-AP; 1:1000 WB), HAUS8 (Abcam, ab95970; 1:250 WB), CAV1 (Proteintech, 16447-1-AP; 1:1000 WB), CAVIN1 (Proteintech, 18892-1-AP; 1:1000 WB), TBC1D13 (ThermoFisher, PA5-61110; 1:2000 WB, 1:200 IF), ARHGAP1 (Proteintech, 11169-1-AP; 1:1000 WB, 1:100 IF), VAMP3 (Proteintech, 10702-1-AP; 1:1000 WB, 1:200 IF), NF1 (Proteintech, 27249-1-AP, 1:1000 WB), ARFGEF1 (Abcam, A44423,1:1000 WB), TBC1D4 (Proteintech, 68063), KIDINS220 (Proteintech, 21856-1-AP, 1:1000 WB), ATP7A (Santa Cruz, sc-376467, 1:1000 WB), CTR1 (Abcam, ab129067, 1:1000 WB), LAT1 (Cell Signaling, 5347s, 1:1000 WB), TBC1D5 (Abcam, 203896, 1:1000 WB), VARP/ANKRD27 (Proteintech, 24034-1-AP, 1:1000 WB), RAB10 (Abcam, ab237703, 1:1000 WB, 1:200 IF).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Expressing, Mutagenesis, Quantitation Assay

Journal: bioRxiv

Article Title: Mapping the endosomal proximity proteome reveals Retromer as a hub for RAB GTPase regulation

doi: 10.1101/2024.11.22.622898

Figure Lengend Snippet: (A-G) Predicted alignment error (PAE) plots and pLDDT structural representation for all seven AlphaFold2 predictions of VPS35:VPS29 association with (A) TBC1D1, (B) TBC1D4, (C) TBC1D5, (D) TBC1D13, (E) DENND4A, (F) DENND4C, and (G) DENND11. (H) GFP based co-immunoprecipitation (co-IP) of GFP-VPS29 wild-type (WT) and hydrophobic pocket mutants after transient transfection in HEK293T cells. Here we blotted for TBC1D4, which contrary to DENND4A/C or TBC1D13 show weak interaction under GFP-VPS29 co-IP.

Article Snippet: Primary antibodies: β-Actin (Sigma-Aldrich, A1978; 1:5000 WB, 1:1000 IF), GAPDH (Sigma-Aldrich, G9545; 1:5000 WB), GAPDH (Sigma-Aldrich, G8795; 1:5000 WB), EEA1 (Cell Signalling; 3288S; 1:200 IF), GFP (Roche; 11814460001; clones 7.1/13.1; 1:1000 WB), mCherry/RFP (Abcam, 167453; 1:1000 WB), FLAG-M2 (Sigma, F1804; 1:1000 WB, 1:200 IF), GLUT1 (Abcam; ab115730; 1:1000 WB; 1:400 IF), LAMP1 (Developmental Studies Hybridoma Bank; AB_2296838; clone H4A3; 1:400 IF), VPS29 (Santa Cruz; D-1; sc-398874; 1:500 WB), VPS35 (Abcam, ab97545; 1:1000 WB, 1:400 IF), VPS35 ( Antibodies.com , A83699; 1:400 IF), VPS26 (Abcam, ab23892; 1:1000 WB), VPS35L (Abcam, ab97889; 1:1000 WB), VPS26C (Sigma-Aldrich, ABN87; 1:1000 WB), VPS35L (Invitrogen, PA5-28553; 1:200 IF), SNX1 (BD Transduction Lab, 611482; 1:1000 WB, 1:200 IF), SNX1 (Proteintech, 10304-1-AP; 1:200 IF), SNX2 (BD Transduction Lab, 611308; 1:1000 WB), SNX5 (Abcam, ab180520; 1:1000 WB), SNX6 (Santa Cruz, sc-365965; 1:1000 WB), SNX17 (Proteintech, 10275-1-AP; 1:1000 WB), SNX17 (Sigma, HPA043867, 1:100 IF), SNX27 (Proteintech, 16329-1-AP; 1:1000 WB), SNX27 (Abcam, ab77799; 1:100 IF), HRS/HGS (Enzo Life Sciences, ALX-804-382-C050; 1:2000 WB, 1:100 IF), STAM (Proteintech, 12434-1-AP; 1:2000 WB, 1:100 IF), FAM21 (Gift from Dan Billadeau; 1:2000 WB), Integrin-α5 (Abcam, ab150361; 1:1000 WB, 1;200 IF), DENND4C (Sigma-Aldrich, HPA014917, 1:1000 WB, 1:500 IF), DENND4C (StressMarq Bioscience, SMC-610, 1:100 IF), DENND4A (Abcam, ab117758, 1:500 WB), HAUS2 (ThermoFisher, PA5-31258; 1:500 WB), HAUS6 (Proteintech, 16933-1-AP; 1:1000 WB), HAUS8 (Abcam, ab95970; 1:250 WB), CAV1 (Proteintech, 16447-1-AP; 1:1000 WB), CAVIN1 (Proteintech, 18892-1-AP; 1:1000 WB), TBC1D13 (ThermoFisher, PA5-61110; 1:2000 WB, 1:200 IF), ARHGAP1 (Proteintech, 11169-1-AP; 1:1000 WB, 1:100 IF), VAMP3 (Proteintech, 10702-1-AP; 1:1000 WB, 1:200 IF), NF1 (Proteintech, 27249-1-AP, 1:1000 WB), ARFGEF1 (Abcam, A44423,1:1000 WB), TBC1D4 (Proteintech, 68063), KIDINS220 (Proteintech, 21856-1-AP, 1:1000 WB), ATP7A (Santa Cruz, sc-376467, 1:1000 WB), CTR1 (Abcam, ab129067, 1:1000 WB), LAT1 (Cell Signaling, 5347s, 1:1000 WB), TBC1D5 (Abcam, 203896, 1:1000 WB), VARP/ANKRD27 (Proteintech, 24034-1-AP, 1:1000 WB), RAB10 (Abcam, ab237703, 1:1000 WB, 1:200 IF).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection

Journal: bioRxiv

Article Title: Mapping the endosomal proximity proteome reveals Retromer as a hub for RAB GTPase regulation

doi: 10.1101/2024.11.22.622898

Figure Lengend Snippet: (A) Confidence level predictor of homo- and heterodimer formation with a network of Retromer binding TBC and DENND proteins. (B) First three ranked PAE plots of the predicted formation of a TBC1D5 homodimer through carboxy-terminal coiled-coil interactions. (C) First three ranked PAE plots of the predicted formation of TBC1D1 homodimer, TBC1D4 homodimer, full length TBC1D1 and TBC1D4 heterodimer, and heterodimer of the carboxy-terminal regions of TBC1D1 and TBC1D4. (D) pLDDT levels depicted on the predicted AlphaFold model of the coiled-coil carboxy-terminal TBC1D1 and TBC1D4 heterodimer.

Article Snippet: Primary antibodies: β-Actin (Sigma-Aldrich, A1978; 1:5000 WB, 1:1000 IF), GAPDH (Sigma-Aldrich, G9545; 1:5000 WB), GAPDH (Sigma-Aldrich, G8795; 1:5000 WB), EEA1 (Cell Signalling; 3288S; 1:200 IF), GFP (Roche; 11814460001; clones 7.1/13.1; 1:1000 WB), mCherry/RFP (Abcam, 167453; 1:1000 WB), FLAG-M2 (Sigma, F1804; 1:1000 WB, 1:200 IF), GLUT1 (Abcam; ab115730; 1:1000 WB; 1:400 IF), LAMP1 (Developmental Studies Hybridoma Bank; AB_2296838; clone H4A3; 1:400 IF), VPS29 (Santa Cruz; D-1; sc-398874; 1:500 WB), VPS35 (Abcam, ab97545; 1:1000 WB, 1:400 IF), VPS35 ( Antibodies.com , A83699; 1:400 IF), VPS26 (Abcam, ab23892; 1:1000 WB), VPS35L (Abcam, ab97889; 1:1000 WB), VPS26C (Sigma-Aldrich, ABN87; 1:1000 WB), VPS35L (Invitrogen, PA5-28553; 1:200 IF), SNX1 (BD Transduction Lab, 611482; 1:1000 WB, 1:200 IF), SNX1 (Proteintech, 10304-1-AP; 1:200 IF), SNX2 (BD Transduction Lab, 611308; 1:1000 WB), SNX5 (Abcam, ab180520; 1:1000 WB), SNX6 (Santa Cruz, sc-365965; 1:1000 WB), SNX17 (Proteintech, 10275-1-AP; 1:1000 WB), SNX17 (Sigma, HPA043867, 1:100 IF), SNX27 (Proteintech, 16329-1-AP; 1:1000 WB), SNX27 (Abcam, ab77799; 1:100 IF), HRS/HGS (Enzo Life Sciences, ALX-804-382-C050; 1:2000 WB, 1:100 IF), STAM (Proteintech, 12434-1-AP; 1:2000 WB, 1:100 IF), FAM21 (Gift from Dan Billadeau; 1:2000 WB), Integrin-α5 (Abcam, ab150361; 1:1000 WB, 1;200 IF), DENND4C (Sigma-Aldrich, HPA014917, 1:1000 WB, 1:500 IF), DENND4C (StressMarq Bioscience, SMC-610, 1:100 IF), DENND4A (Abcam, ab117758, 1:500 WB), HAUS2 (ThermoFisher, PA5-31258; 1:500 WB), HAUS6 (Proteintech, 16933-1-AP; 1:1000 WB), HAUS8 (Abcam, ab95970; 1:250 WB), CAV1 (Proteintech, 16447-1-AP; 1:1000 WB), CAVIN1 (Proteintech, 18892-1-AP; 1:1000 WB), TBC1D13 (ThermoFisher, PA5-61110; 1:2000 WB, 1:200 IF), ARHGAP1 (Proteintech, 11169-1-AP; 1:1000 WB, 1:100 IF), VAMP3 (Proteintech, 10702-1-AP; 1:1000 WB, 1:200 IF), NF1 (Proteintech, 27249-1-AP, 1:1000 WB), ARFGEF1 (Abcam, A44423,1:1000 WB), TBC1D4 (Proteintech, 68063), KIDINS220 (Proteintech, 21856-1-AP, 1:1000 WB), ATP7A (Santa Cruz, sc-376467, 1:1000 WB), CTR1 (Abcam, ab129067, 1:1000 WB), LAT1 (Cell Signaling, 5347s, 1:1000 WB), TBC1D5 (Abcam, 203896, 1:1000 WB), VARP/ANKRD27 (Proteintech, 24034-1-AP, 1:1000 WB), RAB10 (Abcam, ab237703, 1:1000 WB, 1:200 IF).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans

doi: 10.1126/sciadv.adq4461

Figure Lengend Snippet: ( A ) Schematic overview of the major signaling events modulating insulin-stimulated glucose uptake in human skeletal muscle. ( B ) Proximal insulin signaling, as determined by phosphorylation of Akt2 on Thr308 and Ser473 in whole-muscle homogenates. ( C and D ) Distal insulin signaling, as determined by phosphorylation of the Akt substrates GSK3β on Ser9 (C) and TBC1D4 on Thr642 (D) in whole-muscle homogenates. ( E ) GLUT4 translocation, as determined by GLUT4 protein abundance in plasma membrane protein fractions. In-gel stain-free technology was used as a loading control. Lipid, n = 8 (Pre-clamp) and n = 9 (End-clamp); Lipid + mtAO, n = 10. Representative blots ( n = 2 biological replicates from each muscle biopsy sample for each patient). ( F ) Purity of the isolated plasma membrane protein fractions (used to determine GLUT4 translocation), as determined by immunoblot analysis of actin (cytosolic protein marker) and Na + /K + -ATPase subunit α1 (plasma membrane protein marker) in plasma membrane homogenates (PM) as compared with the corresponding whole-muscle homogenates (WM). PM and WM samples were obtained by pooling a given volume of each individual sample. ( G ) Pearson’s correlation between mtAO-induced changes in plasma membrane GLUT4 and leg glucose uptake under insulin stimulation. n = 9. Data [(B) to (E)] presented as observed individual values with estimated means ±95% confidence limits. Linear mixed models were used to estimate within- and between-treatment differences at End-clamp [(B) to (E)]. *Different from Pre-clamp ( P < 0.05). n = 10, unless otherwise stated. Illustrations in (A) were created with BioRender.com .

Article Snippet: The following antibodies were used: Phospho-Akt (Thr308) (Cell Signaling, catalog no. 9275); Phospho-Akt (Ser473) (Cell Signaling, catalog no. 9271); Akt2 (Cell Signaling, catalog no. 3063); Phospho-GSK-3α/β (Ser21/9) (Cell Signaling, catalog no. 9331); GSK-3β (BD Transduction Laboratories, catalog no. 610202); Phospho-TBC1D4 (Thr642) (Cell Signaling, catalog no. 8881); TBC1D4 (Abcam, catalog no. ab189890); GLUT4 (Thermo Fisher Scientific, catalog no. PA1-1065); Na + /K + -adenosine triphosphatase (ATPase) subunit α1 (DSHB, catalog no. α6F); Actin (Sigma-Aldrich, catalog no. A2066); Myc-Tag (Cell Signaling, catalog no. 2272); PRDX2 (Abcam, catalog no. ab109367); PRDX3 (Abcam, catalog no. ab73349); PRDX-SO 2/3 (Abcam, catalog no. ab16830); 4-HNE (Alpha Diagnostics, catalog no. HNE11-s); Goat Anti-Rabbit Ig, Human ads-HRP (SouthernBiotech, catalog no. 4010-05); and Goat Anti-Mouse Immunoglobulins/HRP (Agilent Technologies, catalog no. P0447).

Techniques: Phospho-proteomics, Translocation Assay, Quantitative Proteomics, Clinical Proteomics, Membrane, Staining, Control, Isolation, Western Blot, Marker

Journal: Molecular Metabolism

Article Title: Picalm , a novel regulator of GLUT4-trafficking in adipose tissue

doi: 10.1016/j.molmet.2024.102014

Figure Lengend Snippet: Picalm knockdown in 3T3-L1 cells increase insulin-stimulated GLUT4-translocation . (A) 3T3-L1 adipocytes were treated with siRNA targeting Picalm or with non-targeting siRNA (control). Results of Picalm RT-qPCR verified a successful knockdown. ∗∗p < 0.01 by two-tailed unpaired t-test with Welch correction. (B) 3T3-L1 adipocytes were stimulated with 0, 0.5 or 100 nM insulin for 20 min and subsequently, non-permeabilized cells were stained for plasma membrane GLUT4 (pmGLUT4) and plasma membrane transferrin receptor (pmTfR). Presented are representative pictures of brightfield, digital phase contrast (DPC), and confocal images. (C) Quantification of pmTfR levels at indicated insulin levels and (D) quantification of pmGLUT4 shown for control and si Picalm cells. (E) Western blot analysis of total GLUT4 in control and si Picalm 3T3-L1 cells normalized to Gapdh. ns: not significant by unpaired t-test with Welch correction. (F) Representative images and (G) quantification of western blot analysis of Akt and Tbc1d4 phosphorylation in 3T3-L1 cells with and without Picalm knockdown under basal conditions and after insulin stimulation (0.5 nM (+) or 100 nM (++) for 20 min). Relative values are shown, normalized to the mean of control cells treated with 0.5 nM insulin. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by two-way ANOVA with Sidak's multiple comparison test (C,D) or unpaired t-test with Welch correction (F).

Article Snippet: Primary antibodies recognized Akt (Cell Signaling Technologies, #2920, USA), phospho-T308 Akt (Cell Signaling Technologies, #13038), Akt phospho-S473 Akt (Cell Signaling Technologies, #4060), phospho-T642 Tbc1d4 (Cell Signaling Technologies, #8821), Gapdh (Cell Signaling Technologies, #2118), and GLUT4 (polyclonal antibody recognizing the C-terminus of GLUT4 provided by Professor Geoff Holman and Professor Francoise Koumanov from University of Bath).

Techniques: Knockdown, Translocation Assay, Control, Quantitative RT-PCR, Two Tailed Test, Staining, Clinical Proteomics, Membrane, Western Blot, Phospho-proteomics, Comparison

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Effect of resveratrol on insulin action in primary myotubes from lean individuals and individuals with severe obesity

doi: 10.1152/ajpendo.00299.2023

Figure Lengend Snippet: Signatures on TBC1D4 phosphorylation in the absence or presence of 100 nM insulin in myotubes from lean and subjects with severe obesity in response to 24 h of resveratrol treatment (Resv). A : Thr642 phosphorylation. B : Ser704 phosphorylation. C : Ser341 phosphorylation. D : Ser588 phosphorylation. E : Ser318 phosphorylation. Following detecting significance for either group, treatment, and/or interaction effect using three-way ANOVA, distinguishing meaningful differences were performed using a two-tailed Student’s t test. There were main effects ( P < 0.05) for insulin for all measured sites of pTBC1D4s and for resveratrol for pThr642. There was a group effect with resveratrol for pThr642 and pSer704. Data are presented as means ± SE. n = 8/group (all females); * P < 0.05 for selected comparison. CTR, control.

Article Snippet: Primary antibodies were IRS1(Tyr632) (1:500, 09–433; Millipore, Billerica, MA); IRS1 protein (1:500, sc-559; Santa Cruz Biotechnology, Dallas, TX); Akt (Ser473) (1:1,000, 9271; Cell Signaling); Akt protein (1:1,000, 9272; Cell Signaling); AMP-activated protein kinase (AMPK) (Thr172) (1:1,000, 2531; Cell Signaling); AMPK protein (1:1,000, 2532; Cell Signaling); Akt-substrate at 160 kDa (AS160/TBC1D4) (Thr642) (1:1,000, ab59173; Abcam, Cambridge, MA); AS160/TBC1D4 (Ser588, Ser318, Ser341, Ser704) (1:1,000, customized by Capra Science, Sweden); AS160/TBC1D4 protein (1:1,000, 07-741; Millipore, Billerica, MA); beta actin (1:1,000, 926-42210; LI-COR Biosciences, Lincoln, NE); GLUT4 (1:1,000, sc-53566; Millipore); GLUT1 (1:1,000, Santa Cruz Biotechnology); peroxisome proliferator-activated receptor γ coactivator α (1:1,000, PGC1α) (ab106814; Abcam); SIRT1 (D739) (1:1,000, 2493; Cell Signaling); and citrate synthase (1:1,000, ab96600; Abcam).

Techniques: Two Tailed Test, Comparison, Control

Journal: Journal of cell science

Article Title: Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

doi: 10.1242/jcs.261454

Figure Lengend Snippet: Fig. 2. TBC1D4 and Rab10 are required for AMPK regulation of GLUT4. (A) PM HA–GLUT4–GFP in day 3-differentiated SKM-CRISPR-Cas9 parental and TBC1D4 knockout (KO) cells stably expressing HA–GLUT4–GFP in basal (unstimulated) conditions. Each symbol is the mean calculated from at least 40 cells in individual experiments (n=16). Data are presented as mean±s.e.m. P-value calculated using a paired, two-tailed Student’s two-sample t-test on log-transformed data. (B) Exocytosis assay for HA–GLUT4–GFP under basal (unstimulated) conditions in day 3-differentiated SKM-CRISPR-Cas9 parental and TBC1D4 KO cells stably expressing HA–GLUT4–GFP. Data are cell-associated anti-HA as a function of incubation time in medium containing anti-HA antibody, normalized to the average of the 70, 120 and 150 min of basal condition in each experiment. Data are mean±s.e.m., n=4. Lines show the fit to a single exponential rise to a plateau. (C) Exocytosis rate constants (Ke), expressed as mean±s.e.m., determined from assays shown in B. n=4. P-value calculated using a two-tailed Welch’s two-sample t-test. (D) PM HA–GLUT4–GFP in day 3-differentiated SKM-CRISPR-Cas9 parental cells and Rab10 KO cells stably expressing HA–GLUT4–GFP, treated with or without 3 µM PF739 for 60 min. Each symbol is the mean value of at least 40 cells per experiment (n=8). Data are presented as mean±s.e.m., normalized to the basal condition of parental cells. P-value two-way ANOVA with unequal variances; P<0.0001; pairwise P-values in the figure have undergone FDR multiple comparison adjustment. (E) Exocytosis assay for HA–GLUT4–GFP under PF739-stimulated conditions in day 3-differentiated SKM parental and Rab10 (R10) KO cells stably expressing HA–GLUT4–GFP. Data are cell-associated anti-HA as a function of incubation time in medium containing anti-HA antibody, normalized to the average of 70, 120 and 150 min of control condition in each experiment. Data are mean±s.e.m., n=4. Lines show the fit to a single exponential rise to a plateau. (F) Endocytosis assay for HA–GLUT4–GFP in day 3-differentiated SKM- CRISPR-Cas9 parental cells and Rab10 KO cells stably expressing GLUT4 treated with or without 3 µM PF739 for 60 min. Data are a ratio of internal anti-HA (internalized pulse of GLUT4) to PM GLUT4. Data are normalized to the 6 min time point of basal (unstimulated) condition in each experiment. Mean±s.e.m., n=3. Lines show the fit to a straight line. A.U., arbitrary units.

Article Snippet: Primary antibodies used for western blotting were against β-tubulin (1:2000; ab6046, Abcam), Rab10 (1:500; 4262S, Cell Signaling Technology), Rab8A (1:1000; 610844, BD Biosciences), myosin heavy chain (1:1000; M4276, Millipore Sigma), myogenin (1:1000; ab1835, Abcam), total acetyl-CoA carboxylase (1:1000; 3676, Cell Signaling Technology), labelled-acetylCoA carboxylase (Ser79 ACC) (1:500; 3661, Cell Signaling Technology), labelled T308-AKT (1:500; 2965S, Cell Signaling Technology), total AKT (1:1000; 9272S, Cell Signaling Technology), labelled-T642 TBC1D4 (1:1000; 8881S, Cell Signaling Technology) and total TBC1D4 (1:1000; 07741, Millipore Sigma).

Techniques: CRISPR, Knock-Out, Stable Transfection, Expressing, Two Tailed Test, Transformation Assay, Incubation, Comparison, Control, Endocytosis Assay