Pel h2ax s139 t

The following commercial antibodies were used in the Western blot experiments: anti-p21 (WAF1, Cip1) (#14-6715-81, Invitrogen, Carlsbad, CA, USA), anti-p16INK4α (#PA5-20379, Invitrogen, Carlsbad, CA, USA), anti-p53 (#ab131442, abcam, Cambridge, MA, USA), anti-β actin (#A5441, Sigma-Aldrich, St. Louis, MO, USA), anti-Mfn1 (#14739, Cell Signaling Technology, Danvers, MA, USA), anti-Mfn2 (#9482, Cell Signaling Technology, Danvers, MA, USA), anti-Opa1 (#612606, BD Biosciences, San Jose, CA, USA), anti-VDAC (#4661, Cell Signaling Technology, Danvers, MA, USA), anti-Drp1 (#611112, BD Biosciences, San Jose, CA, USA), anti-GAPDH (#ABS16, Sigma-Aldrich, St. Louis, MO, USA), Ser139_phospho detection antibody H2AX (#PEL-H2AX-S139-T, RayBiotech, Norcross, GA, USA), pan detection antibody H2AX (#PEL-H2AX-S139-T, RayBiotech, Norcross, GA, USA).

The HMEC-1 cells were irradiated with different doses of IR (0 Gy, 0.25 Gy, and 8 Gy) and then lysed to obtain cell lysates. To measure the levels of phospho-H2AX and total H2AX in the lysates at 1 h after IR, ELISA was performed using the RayBio^® phospho-H2AX (Ser139) and total H2AX ELISA kit (#PEL-H2AX-S139-T, RayBiotech, Norcross, GA, USA), following the manufacturer’s instructions. Western blot analysis was also performed using the same detection antibodies provided in the ELISA kit to confirm the ELISA results. To standardize the results for both ELISA and Western blot analysis, the absorbance values at 450 nm for phospho-H2AX in ELISA and the band intensity for phospho-H2AX in Western blot analysis were adjusted by dividing them with the optical density values and band densimetry values for pan-H2AX, respectively. The resulting values were expressed as rH2AX/H2AX for each sample. The sample size was N = 4 per radiation dose. The results were analyzed using one-way ANOVA and Dunnett’s multiple comparisons test, with a significance level of * p < 0.05.