Appropriate horseradish peroxide conjugated secondary antibodies

Western blot was performed as described previously (19 (link), 20 (link)), using pATM (Ser1981) (D6H9), MRE11, RAD50, NBS1, PARP-1, pAKT pCHK2 (Thr68) (C13C1), γH2AX (Cell Signaling, Danvers, MA), and β-Actin (8H10D10) antibodies (Cell Signaling). Appropriate horseradish peroxide-conjugated secondary antibodies (Cell Signaling) were used, and proteins were detected with the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA). The blotted membranes were stripped and re-probed with ATM (D2E2), AKT, and CHK2 (D9C6) antibodies (Cell Signaling). Protein bands were captured and quantified by Chemi Doc^TM MP Imaging System (Bio-Rad).

Naive CD4 T cells purified from HCV-infected individuals and HS were lysed on ice in RIPA lysis buffer (Boston BioProducts Inc, Ashland, MA) in the presence of protease inhibitors (Thermo Scientific, Rockford, IL). The protein concentrations were measured by Pierce BCA protein assay kit (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat milk, 0.5% Tween-20 in Tris buffered saline, and incubated with the pATM (Ser1981) (D6H9), pBRCA1, pCHK1, and pCHK2 (Thr68) (C13C1) antibodies and β-Actin (8H10D10) antibodies (Cell Signaling, Danvers, MA). Appropriate horseradish peroxide-conjugated secondary antibodies (Cell Signaling) was then used and proteins were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA). Membranes were stripped and re-probed with MER11, RAD50, NBS1, BRCA1, ATM (D2E2), γH2AX, PARP, CHK1, and CHK2 (D9C6) antibodies (Cell Signaling). Protein bands were captured and quantitatively analyzed by Chemi DocTM MP Imaging System (Bio-Rad System).

Naïve CD4 T cells were treated with 5 μM KML001 or DPBS control for 48 h. The cells were harvested and lysed on ice in RIPA buffer (Boston BioProducts Inc, Ashland, MA) in the presence of protease inhibitors (Thermo Scientific, Rockford, IL). The protein concentrations were measured by Pierce BCA protein assay kit (Thermo Scientific). The proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, pre-blocked with 5% non-fat milk, 0.1% Tween-20 in Tris buffered saline (TBS), and incubated with the following primary antibodies: PARP1, TRF1, TRF2, POT1, TIN2, RAP1, TPP1, P53, ATM, pATM, Top1, Top2a, Apollo, MRE11, RAD50, NBS1, or Sirt6. The membranes were reprobed with β-Actin antibody (Cell Signaling, Danvers, MA) as a loading control. P24 antibody was obtained from NIH/NIAID AIDS reagent program. Appropriate horseradish peroxide-conjugated secondary antibodies (Cell Signaling) were used, and the proteins were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA). Protein bands were captured and quantified using the Chemi DocTM MP Imaging System (Bio-Rad System).