Figure 1
From: TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes
Identification and validation of the TBC1D1 PTB protein interactome. (a) Schematic of the SILAC experiment setup used to identify proteins interacting with the PTB1 and PTB2 domains (amino acids 1–381) of TBC1D1 in differentiated C2C12 myotubes. (b) Table of proteomics output filtered for a score > 30, enrichment > 4 and #PSMs > 8 (raw dataset is in Supplementary Table S1). Shown in italics are the previous reported hits33. Score: the sum of the Xcorr values for the peptides matched to that protein (a high score generally signifies a high protein abundance and a high confidence in the detection and quantification by the software). Enrichment: Ratio of the quantification values of the medium (GFP-PTB1 + 2) and light (GFP) quantification channels. A ratio of 100 suggests that the protein was not detected at all in the light (GFP) channel. Coverage: percentage of the protein sequence covered by identified peptides. # PSMs (peptide spectrum matches): total number of identified peptide sequences (includes those redundantly identified). # Peptides: total number of unique identified peptides. (c) Western blot validation of proteomics results. GFP-trap precipitates from C2C12 myotube extracts, lentivirally transduced to express either GFP or GFP-PTB1 + 2 blotted for the proteins indicated. (d) Lysates from differentiated C2C12 myotubes lentivirally transduced with GFP or GFP-TBC1D1 subjected to GFP-trap and western blotting for the proteins indicated. (e) Homogenised quadriceps lysates from either wild type (WT) mice or mice expressing 3xFLAG-TBC1D1 were immunoprecipitated with an anti-FLAG antibody and resulting complexes analysed. Representative blots of n = 4 independent experiments.
