Bp9703 100

To generate transient KO cells of the different genes identified by mass spectrometry, guide RNAs targeting the gene of interest were cloned into pKLV-U6gRNA (BbsI)-PGKblast2ABFP. Viral particles were produced with Lenti-X 293T cells as described above. The HeLa Cas9 stable cell line was then transduced. Briefly, 1 × 10⁴ HeLa Cas9 cells were infected with 250 µl of lentiviral supernatant in a 96-well plate. Seventy-two hours posttransduction cells were replated in a 48-well plate and incubated for an extra 2 d. On d 6, 2 × 10⁴ of transduced cells were plated onto Matrigel-coated (1:100 in complete DMEM; 354277; Corning) glass coverslips (400-03-19; Academy). On d 8, cells were fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.1% TX-100 (T9284; Sigma-Aldrich) and incubated for 1 h with a blocking solution containing 3% BSA (BP9703-100; Sigma-Aldrich). Cells were incubated for 1 h with anti-ACBD3 and anti-GM130 primary antibodies followed by 1 h incubation with appropriate Alexa Fluor Dyes. PBS1X was used to wash cells in-between all steps and DAPI stained (300 nM; D21490 Invitrogen) for 5 min. Coverslips were mounted in ProLong Gold (P36930; Life Technologies).