Mouse anti β tubulin antibody

Cells were lysed with RIPA buffer (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific) and sonicated for 30 seconds. Lysates were separated by SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and analyzed by immunoblotting. Rabbit anti-CPT1A (No. 12252) (Cell Signaling Technology) was used as a primary antibody. Immunoblotting for β-tubulin by mouse anti-β-tubulin antibody (Santa Cruz Biotechnology) and COX IV by rabbit anti-COX IV antibody (Cell Signaling) was used as a loading control for whole-cell lysates and mitochondrial lysates, respectively. Anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology and Thermo Fisher Scientific, respectively. Signal was detected using the ECL system with X-ray film development (Thermo Fisher Scientific and GE Healthcare Life Sciences) or a LI-COR C-Digit blot scanner (LI-COR) according to the manufacturer’s instructions.

Cell lysates were prepared by adding 100 µL of M-PER extraction buffer (Thermo Scientific, Waltham, MA, USA) per 1 × 10⁶ cells. The total protein concentration in cell lysates or purified virus samples was determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific). 15 µg of protein extracts or purified virus like particles were separated in a 4–12% (w/v) acrylamide NuPAGE gradient pre-cast gel (LifeTechnologies). Samples were resolved for 40 min at a constant voltage of 180 V and transferred into a PVDF membrane (Merck, Billerica, Massachusetts, EUA) using a Trans-Blot^® Turbo™ Transfer System (BioRad, California, USA). After transferring, PVDF membranes were blocked with 4% (w/v) skimmed milk (Merck) and incubated with the respective primary antibody: rabbit anti-CD81 (Sigma-Aldrich), mouse anti-β-Tubulin antibody (SantaCruz, CA, USA), mouse anti-HCV E1 (Acris Antibodies, Herford, Germany), mouse anti-HCV E2 (Austral Biologicals), mouse anti-HCV Core (SantaCruz) or the anti-MLV p30 monoclonal antibody produced by the hybridoma R187 (ATCC). Detection was performed with the corresponding anti-mouse or anti-rabbit secondary antibody conjugated to Horseradish peroxidase and developed using the ECL Detection Reagent (GE Healthcare, Chicago, IL USA).

Primary antibodies: anti-β-actin antibody (Cat# A5316-100), rabbit anti-katanin p60 AL2 antibody (N-15) (Cat# sc-84855), goat anti-katanin p60 A1 antibody (M-13) (Cat# sc-10929), mouse anti-katanin p60 AL1 antibody (A-10) (Cat# sc-373814), rabbit anti-fidgetin antibody (H-146) (Cat# sc-68343), goat anti-FIGNL1 antibody (C-12) (Cat# sc-138278), goat anti-FIGNL2 antibody (G-14) (Cat# sc-242820), mouse anti-β-tubulin antibody (Cat# sc-5274), and rabbit anti-ZP3 antibody (H-300) (Cat# sc-25802) were purchased from Santa Cruz (USA); mouse anti-spastin antibody (Cat# S7074) was purchased from Sigma (USA); mouse anti-5-methylcytosine antibody (Cat# ab73938) was purchased from Abcam (England). Another rabbit anti-fidgetin antibody was custom-made (antigen sequence: GLTPIAPSALTNNSA) and purified by Zoonbio Biotechnology (China).
Secondary antibodies: HRP-conjugated anti-rabbit IgG and anti-mouse IgG (Vazyme, China), Cy2-conjugated anti-mouse IgG (Cat# 715-225-150), Cy2-conjugated anti-rabbit IgG (Cat# 715-225-152), rhodamine-conjugated anti-mouse IgG (Cat# 715-025-150), all purchased from Jackson ImmunoResearch Laboratory (USA).