West pico chemiluminescent staining

Samples were normalized by protein concentration with PBS, mixed 1:1 with 2x SDS-PAGE loading buffer, boiled for 10 min., separated by SDS-PAGE, and transferred to PVDF. Proteins were detected by western blot using the following primary antibodies: Anti-CypA (C-14), anti-CypB (k2E2), anti-ERGIC (H-245), anti-actin (C4), and anti-GAPDH (6C5) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-Topo IIa was from BD Biosciences (San Jose, CA). The HRP-conjugated anti-Flag (M2) antibody was from Sigma-Aldrich (St Louis, MO). HRP-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were also from Santa Cruz Biotechnology. Antibody binding was detected and visualized by West Pico chemiluminescent staining (Thermo Scientific, Rockford, IL). Images were captured by exposure to radiographic film, scanned to computer, adjusted for brightness and contrast if necessary, and cropped for size.

Protein were normalized to equal concentrations with PBS and mixed 1:1 with 2x SDS-PAGE loading buffer and boiled for 5 min. Samples (20 μg/lane) separated by SDS-PAGE using Tris-glycine gels, and transferred to PVDF. Proteins were detected by immunoblot using the following primary antibodies: anti-HIV-1 p24 (Toohey et al., 1995 (link); Wehrly and Chesebro, 1997 (link)), anti-ERGIC (H-245), Actin (C-4), Sumo2/3 (N-18), GAPDH (6C5), EEF1A1 (CBP-KK1), hnRNP-L (4D11), NONO (H-85), PCNA (PC10), and the HRP conjugated anti-rabbit, anti-mouse, and anti-human IgG secondary antibodies were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AURKA was obtained from Cell Signaling Technology (Danvers, MA). Anti-PPP2R5D was obtained from Abcam (Cambridge, MA). HRP conjugated anti-Flag (M2) primary antibody was from Sigma-Aldrich (St Louis, MO). All antibodies were diluted in blot wash buffer (TBS with 0.4% tween-20). HRP secondary antibodies were detected using West Pico chemiluminescent staining (Thermo Fisher Scientific) and exposure to radiographic film. Images were scanned to computer, adjusted for brightness and contrast if necessary, and cropped for size.