Fig 1: Photo-cross-linking of photo-CbA to Sec61α and CbA stabilization of Sec61 in cells. Photocotransin (CT7) or photocoibamide (pCbA) cross-linking to cells or sheep rough microsomes (SRM). Samples were photolyzed and the covalent adduct detected by click-chemistry to TAMRA-azide reporter and in-gel fluorescence scanning and Western blotting. (A) A431 cell lines were first incubated with CbA or carrier, followed by incubation with photo-CbA, photolysis, and click chemistry. Following SDS PAGE, lysates were queried for in gel fluorescence and subsequently transferred for anti-Sec61α and anti-FLAG Western Blot. Arrows indicate Sec61 (with or without a 10 kDa insert), star indicates nonspecific WB signal, triangle indicates free TAMRA dye within the gel. (B) As in A but in SRMs. Sample without UV irradiation shows the nonspecific background labeling of photo-CbA. (C) SRMs were first incubated with indicated concentrations of AprA, or Myco, followed by incubation with photo-CbA, photolysis, and click chemistry. (D) CT7 cross-linking to microsomes in the presence or absence of 10 μM CbA. (E) Stabilization of intracellular Sec61α by CbA. Isothermal concentration–response analysis of Sec61α in the presence or absence of CbA (0.01 nM to 3 μM), OSU-03012 (0.03 nM to 10 μM), or 0.1% DMSO. Intact U87-MG glioblastoma cells were treated as indicated and subjected to heating at 51 °C for 3 min. Heat-treated cell suspensions were snap frozen, lysates cleared by centrifugation, and the soluble fraction analyzed by SDS-PAGE and Western blotting. Note that Sec61α migrated above the 40 kDa molecular weight marker in several human cell lines using a standard Western blot protocol (Abcam ab183046; Figure S26C). (F) Quantification of immunoblot data shown in E. Sec61α band intensity was normalized to tubulin, and data points were fitted using nonlinear regression analysis.