Hpa008320

Tet-ON HeLa shRNA cell lines were treated with Dox for six consecutive days and stressed with 300 nM Tg over a 24-h time course. Cells were harvested after 0, 2, 4, 8, 16 and 24 h and lysed in high salt buffer (20 mM Tris pH 7.5, 400 mM NaCl, 0.5% (v/v) NP-40, 0.3% (v/v) Triton X-100) supplemented with 0.1 mM PMSF (Sigma) and protease inhibitor cocktail (Roche). Protein concentration was determined using Bradford assay (Bio-Rad), and 30 μg of each sample were separated by SDS–PAGE. For B cells, cells were counted, directly lysed in 1× SDS loading buffer at 95°C for 5 min and loaded at 1–2 × 10⁶ cells per lane. Proteins were transferred to Immobilon-P membranes (Millipore) and probed with the following antibodies using standard protocols: lamin A/C (Sigma, 4C11 1:1,000), HSP90 (Abcam, 3A3 1:2,000), calnexin (Santa Cruz, C-20 1:1,000), RTCB (described previously (Popow et al, 2011 (link)), 1:5,000), monoclonal antibody against archease was generated by immunization of mice, fusion of splenocytes and generation of hybridoma (Monoclonal Antibody Facility, Max F. Perutz Laboratories, Vienna 1:500), β-actin (Sigma, A2006 1:5,000), XBP1s (BioLegend, 1:500), DDX1 (Bethyl, A300-521A 1:1,000), FAM98B (Sigma, HPA008320 1:500) and CGI-99 (Sigma, HPA039824 1:1,000).

Western blotting was carried out as described previously [17] (link). The following primary antibodies were used: for hCLE, a rabbit polyclonal antibody (1∶1000) from Abcam; for RNAP II, a monoclonal antibody 8WG16 (1∶500) from Covance; for β-tubulin, a monoclonal antibody (1∶15000) from SIGMA, for DDX-1, a goat polyclonal antibody sc-49817 (1∶200) from Santa Cruz Biotechnology; for HSPC117/C22orf28, a goat polyclonal antibody LS-C139785 (1/3000) from LifeSpan BioSciences; for FAM98B, a rabbit polyclonal antibody HPA008320 (1/500) from Sigma; for α-actin, a rabbit polyclonal antibody (1/200) from Abcam and for S6, a mouse monoclonal antibody 54D2 (1/1000) from Cell Signaling that recognizes total S6 protein.