Pegfp sec23a

Cells were grown overnight in 24-well plates at 37°C in a 5% CO₂ atmosphere. After reaching over 80% confluence, the plasmids mRuby-Clathrin (Addgene #55852), pEGFP-Sec23A (Addgene #66609), ZsGreen-Rab5 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.), EGFP-Rab7A (Addgene #28047), GFP-LAMP1 (Addgene #16290), EMTB-3XGFP (Addgene #26741), pSNAPf-Cox8A (Addgene #101129), or pSNAPf-TOMM20 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.) was transfected into the cells using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 24 h, the transfected cells were digested with trypsin-EDTA and seeded into Nunc Glass Bottom Dishes (Φ 12 mm, Thermo Fisher Scientific, Inc.) at a density of 1.5 ~ 2.0 × 10⁴ per well in growth medium (150 μL). The cells were grown for an additional 12 ~ 24 h before incubation with the indicated probes.

pEGFP-Sec23A (Addgene plasmid # 66609)^{62 (link)} was a gift from Dr. David Stephens. Plasmids encoding mouse and human CCTα, β2, β3, and β3 mutants were cloned by PCR using cDNA of mouse and human cells as templates and tagged with mCherry at the C terminus. Primer sequences for PCR are provided in Supplementary Table 1. Plasmids were transfected to MEFs and U2OS using Lipofectamine 2000 or Lipofectamine 3000 (Thermo Fisher).