Anti golgin97

Protein lysates were prepared for western blot analysis, as described [4 ]. Affinity-purified anti-TMF antibodies (1:1000, Sigma, USA) were used to detect the TMF protein by western blot (WB) analysis. Tubulin, golgin97 and Hexokinase I (HK I) levels were detected using anti-tubulin (DSHB, USA, 1:1000), anti- HK I (Cell Signaling, USA, 1:1000) and anti-golgin97 (Abcam, USA, 1:1000) antibodies.

SDS-PAGE analysis of proteins was performed with 4-12% NuPAGE Bis-Tris gels (Invitrogen) with 3-(N-morpholino) propane sulfonic acid running buffer and staining with Bio-Safe Coomassie G250 (Bio-Rad). Aliquots of ~5 µg of proteins were loaded per gel lane. Proteins in gels were transferred onto polyvinylidene fluoride membranes, blocked with tris-buffered saline (pH 7.4) containing 2% bovine serum albumin, 3% casein or skim milk, and 0.5% Tween 20 at 4°C overnight. They were incubated with primary antibodies at 25°C for 1 h. Primary antibodies tested include anti-CD19 (eBioscience), anti-CD5 (sc-6985, Santa Cruz Biotech), anti-CD72 (sc-25265), anti-CD21 (sc-7027), anti-CD81 (sc-9158), anti-PI3K (sc-31969), anti-Grp78 (Abcam), anti-Golgin97 (Abcam), biotin anti-CD95 (Fas/APO-1, eBioscience), or biotin rat anti-mouse preBCR (BD Biosciences). Membranes were washed thrice with TBS containing 0.5% Tween 20, then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase in blocking buffer at 25°C for 1 h, and developed with enhanced chemiluminescence substrate. Secondary antibodies used include goat anti-rabbit IgG-horseradish peroxidase (HRP), anti-goat IgG-HRP, or streptavidin-HRP (BioLegend).