Hsp90

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).

Proteins were mixed with 5 × Laemmli buffer and incubated at 100 °C for 10 minutes. Samples were immediately transferred in the ice afterwards. All samples and prestained protein ladder (10-250 kDa, Thermo ScientificTM) were loaded into the columns of NuPAGETM 4-12% Bis-Tris Gel (Invitrogen) placed in the Invitrogen tank containing 1× NuPAGE MOPS SDS running buffer (Novex). The gel was run at 100v for 75 minutes and transferred to a PVDF (Polyvinylidene difluoride) membrane (Millipore) using NuPAGE transfer buffer (Novex) with methanol and antioxidant (Invitrogen) at 35v for 60 minutes. The membrane was blocked with 5% milk-TBST at RT for 1 hour and incubated overnight with TRIB1 (Millipore), and HSP90 (Abcam) diluted in 5% milk-TBST (1:1000 and 1:5000 respectively) at 4 °C. The membrane was then washed with 0.1 v/v TBST for 5 minutes 3 times and incubated with Polyclonal Goat anti-Rabbit Immunoglobulin/HRP, and Polyclonal Rabbit anti-Rat Immunoglobulin/HRP (Dako) diluted in 5% milk-TBST (1:2500 and 1:5000 respectively) at RT for 1 hour. The membrane was then washed with TBST 3 times for 5 minutes, incubated with ECL, and imaged with Bio-Rad imager.

Concentrated culture supernatant samples were separated on 12% SDS-PAGE gel, transferring proteins to a nitrocellulose membrane. Primary antibodies against HSP27, HSP70, HSP90 (Abcam, Cambridge, UK) and HRP-labeled goat anti-mouse Ig (Jackson Laboratory, Bar Harbor, ME, USA) secondary antibodies were applied, using ECL reagent (Amersham Biosciences, Piscataway, NJ, USA) for detection.