Dna polymerase theta

A total lysate of 30 μg protein was loaded on SDS-Gels and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies overnight and, subsequently, probed with secondary HRP-conjugated antibodies. Primary antibodies: GAPDH (Meridian Bioscience H86504M, Cincinnati, OH, USA), Ku70 (Santa Cruz Biotechnology sc-1486, Heidelberg, Germany), Ku80 (Santa Cruz Biotechnology sc-9034, Heidelberg, Germany), DNA ligase IV (Santa Cruz Biotechnology sc-28232, Heidelberg, Germany), Mre11 (Cell Signaling 4895, Danvers, MA, USA), DNA ligase III (BD Transduction Laboratories 611876, Franklin Lakes, NJ, USA), PARP (Cell Signaling 9542), DNA polymerase theta (Abcam ab80906, Cambridge, UK).

H&E staining, immunohistochemistry, and alcian blue staining were performed from paraffin-embedded tissue samples; 1–2 µm slides were cut by microtome (Leica). All antibodies for immunohistochemical staining were used in 1:50, 1:100, or 1:200 dilution, and incubated overnight at 4 °C. Primary antibodies: DNA polymerase theta (Abcam ab111218, Cambridge, UK), PARP1 (Abcam ab32138, Cambridge, UK), Mre11 (Cell Signaling 4895, Danvers, MA, USA), Ku70 (Santa Cruz Biotechnology sc-1486, Heidelberg, Germany), Ki67 (Bethyl Laboratories IHC-00375, Montgomery, TX, USA), PCNA (Cell Signaling 2586, Danvers, MA, USA), CyclinD1 (Cell Signaling 2978, Danvers, MA, USA), p-ERK1 (Cell Signaling 4370, Danvers, MA, USA), Cox2 (Cell Signaling 12282, Danvers, MA, USA). Alcian blue was performed by Alcian Blue pH 2.5 Stain Kit (Vector Laboratories H-3501, Newark, CA, USA), according to the manufacturer’s protocol. All slides used for histological analysis were scanned with the Pannoramic Midi II (3DHISTECH, Budapest, Hungary), and evaluated with different magnification using Quant Center software (3DHISTECH, Budapest, Hungary).