Mouse anti gapdh antibody

Rat KIF5A cDNA was cloned into pEGFP-N3 (Clontech) to create construct KIF5A-EGFP. Similarly, mouse KIF5B and KLC1 cDNAs were cloned into the pEGFP-C vector to obtain EGFP-KIF5B and EGFP-KLC1, respectively. pGFP-KIF5C was from Addgene (#71853). Rabbit antibodies anti-KIF5A (1:2000; 21186-1-AP), KIF5B (1:2000; 21632-1-AP), KIF5C (1:2000; 25897-1-AP), and mouse antibody anti-β-actin (1:10,000; 60008-1-Ig) were obtained from Proteintech (Rosemont, IL); rabbit anti-APP antibody (1:5000; A8717; St. Louis, MO) was purchased from MilliporeSigma; mouse anti-GFP antibody (1:2000; sc-9996) and goat anti-KLC1 antibody (1:1000; sc-13362) were from Santa Cruz Biotechnology (Dallas, TX). The mouse anti-GAPDH antibody (1:3000; GTX627408) was from GeneTex (Irvine, CA).

CRFK cells were lysed with 100 µL of reducing SDS sample buffer and heated at 100°C for 10 min. Twenty microliters of the supernatant was loaded onto a 12% SDS polyacrylamide gel and semi-dry transferred onto a PVDF membrane. The membrane was blocked with 5% milk powder in PBS and incubated with either a mouse anti-VP1 antibody (Abcam) or a mouse anti-GAPDH antibody (GeneTex) for 1 h. After washing the membrane, the bound antibodies were detected using an Alexa Fluor 488 labeled goat anti-mouse IgG (Thermo Fisher, diluted 1:5,000). VP1 expression levels were quantified by analyzing the VP1 bands with ImageJ software. The data were normalized by the respective control GAPDH bands and correlated with the quantity at 0 min.

HEK293T cells expressing Cas9 transiently with or without ASV treatment were harvested and lysed in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1× HALT protease inhibitor cocktail (Thermo Fisher Scientific) and 5 mM EDTA. Protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Approximately 20 μg of protein lysate was separated on a NuPAGE 4%–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) with 1× NuPAGE SDS running buffer, transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA), and blotted with a mouse anti-GAPDH antibody (1:2,000, GeneTex, Irvine, CA, USA) or a mouse anti-Cas9 antibody (1:1,000, Novus Biologicals, Littleton, CO, USA). After washing, the membranes were incubated in horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG; 1:5,000, Thermo Fisher Scientific) at room temperature for 1 h, followed by exposure to enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific). Membranes were visualized using a ChemiDoc imaging system (Bio-Rad).