Actin antibodies

Whole cell extracts were prepared from HNSCC cells and analysed by immunoblotting as previously described (9 (link)). RAD51 antibodies were from Bethyl Laboratories (Montgomery, USA), ATR antibodies were from Abcam (Cambridge, UK), CHK1 antibodies were from Cell Signalling Technology (Leiden, The Netherlands), and actin antibodies were from Merck-Sigma (Gillingham, UK). For immunofluorescent staining of RAD51, cells were grown on 13-mm coverslips, unirradiated or irradiated with 4 Gy x-rays, and allowed to repair for 4 h in 5% CO₂ at 37°C, prior to fixing and staining as previously described (9 (link)).

RWPE-1, PC3, DU145, VCaP, 22Rv1 and LNCaP were obtained from American Type Culture Collection (ATCC), that are authenticated by Short Tandem Repeat Profiling and used within 5–10 passages and replenished from stocks. C4-2B is described earlier². All cell lines were incubated in a humidified atmosphere of 5% CO₂ at 37 °C and cultured as described earlier^{2 (link)}. Cultures are routinely tested for Mycoplasma contamination using the MycoAlert™ PLUS Mycoplasma Detection Kit from Lonza and by PCR based screening. HOXB13 F-9 monoclonal antibody (Cat #SC-28333) was purchased from Santa Cruz and HOXB13 rabbit polyclonal antibodies were purchased from Genetex (GTX129245). HSPB8 (Abcam Cat# Ab79784), Actin antibodies (Sigma-Aldrich Cat# A2228), were purchased from Abcam. AR (F39.4.1) antibody was purchased from Biogenex. siRNAs were purchased from multiple sources. Source for all siRNA and sequences are provided in the Supporting data section. GFP-HSPB8 expression vector was synthesized and sequenced by Genscript. Prostate cancer cells were transfected with siRNAs or plasmids using LONZA nucleofector kit.

OPSCC cells (UMSCC6, UMSCC74, UMSCC47) were kindly provided by Prof T. Carey, University of Michigan, USA and were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15 % fetal bovine serum, 2 mM L-glutamine, 1× penicillin-streptomycin and 1× non-essential amino acids. UPCI-SCC090 were kindly provided by Dr S. Gollin from the University of Pittsburgh and were cultured in Minimal Essential Medium (MEM) supplemented with 15 % fetal bovine serum, 2 mM L-glutamine, 1× penicillin-streptomycin and 1× non-essential amino acids. All cells were cultured under standard conditions in 5 % CO₂ at 37°C, and were authenticated in our laboratory by STR profiling. APE1, XRCC1, Pol β and PARP-1 antibodies raised in rabbit and affinity purified were kindly provided by Dr G. Dianov. PNKP antibodies raised in rabbit were kindly provided by Dr M. Weinfeld. DNA-Pk, RAD51, BRCA2, Ku-86 and PAR antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany), 53BP1 antibodies were from Bethyl Laboratories (Montgomery, USA), p16 antibodies were from BD Biosciences (Oxford, UK) and γH2AX antibodies were from Millipore (Watford, UK). Actin antibodies were from Sigma-Aldrich (Gillingham, UK). Goat anti-mouse Alexa Fluor 555 and goat anti-rabbit Alexa Fluor 488 secondary antibodies for immunofluorescence staining were from Life Technologies (Paisley, UK).