Tgx mini gel

Whole cell lysates were made from pelleting 1 × 10⁷ cells at 4C at 3000 x g and resuspending in lysis buffer (20 mM Tris-HCl, pH 8.0; 150 mM KCl; 5 mM MgCl2; 250 mM Sucrose; 1 mM DTT; 10 mM Digitonin; 1 mg/ml Sodium Heparin; 1 mM Pefabloc SC; 0.5 U/μl DNaseI; 1 U/μl SUPERaseIN). Cells were incubated in lysis buffer for 10 min on ice and passed through a 30G needle 5x. Insoluble material was pelleted at 6000 x g for 10 min at 4C. Lysate (1 × 10⁶ cells/sample) was run on a 4–20% TGX mini-gel (Bio-Rad) for 45 min at 200 V and transferred onto 0.2 μm nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad) with semi-dry settings 25V for min. The blot was blocked for 30 min with Odyssey PBS Block (Li-cor). The blot was probed with biotinylated jacalin (1:4,000; Vector Labs) and E7 anti-tubulin antibody (1:10,000; Developmental Studies Hybridoma Bank) diluted in block for 1 hr, and then with IRDye 800 streptavidin (1:1,000; Li-cor) and IRDye 700 mouse (1:1,000; Li-cor) in PBST [PBS with %1 Tween 20 (v/v)]. Blot was imaged on Licor Odyssey (Figure 4—figure supplement 1).

Protein (and crude viral lysates) were mixed at an equal ratio with a 2X reducing sample SDS-PAGE buffer, heated to 75 °C for 20 minutes, separated on a 4–20% precast TGX mini-gel (Biorad), and transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% BSA in TBS with 0.05% Tween (TBST) for 30 minutes, before incubating at 4 °C overnight with agitation with the appropriate antibody diluted in 5% BSA in TBST. Following three washes in TBST for 10 minutes each, membranes were incubated at room temperature with mild agitation with an appropriate secondary HRP antibody (Sigma Aldrich, 1:5000) diluted in 5% BSA in TBST, washed three times with TBST for 10 minutes each, and developed with enhanced chemiluminescent substrate (Amersham-Pharmacia Biotech, USA).