Western blot analysis

Primary HFMs were seeded into 6-well plates (~ 500,000 per well) and left to attach overnight. Prior to treatment, cells were incubated in starved media for 24 h (2:1 melanocyte media minus FCS and BPE). After treatment, media was removed and cells rinsed in PBS before lysis with 200 μl ice-cold RIPA buffer (Sigma, UK) containing protease inhibitors (Complete EDTA protease inhibitor tablets; Roche, UK) and phosphatase inhibitors (phosphatase inhibitor tablets; Pierce, UK). Cell lysates were sonicated briefly for 15 s then protein concentration measured using the Microplate BCA. Protein Assay Determination Kit—Reducing Agent Compatible (Pierce, UK). Lysates were diluted in 2 × Laemelli SDS sample buffer containing 50 mM DTT to equal concentration then ~ 10 μg of protein electrophoresed through NuPage 3–8% Tris–Acetate gels (Thermofisher Scientific) alongside Kaleidoscope (Bio-Rad, UK) and HiMark (Invitrogen, UK) protein standards. Protein was transferred to PVDF membrane (Mini Trans-blot Turbo Transfer Pack) using the Trans-blot Turbo module at 2.5 A constant (~ 25 V) for 30 min. PVDF membranes were cut to size and blocked in 5% (w/v) non-fat milk powder dissolved in TBS-T (TBS-Tween 20 (0.01% v/v)) for at least 1 h at room temperature (RT). Membranes were incubated in either Abcam Cat# ab32420 Rabbit anti-ATM [Y170] antibody diluted 1:4000 in 5% milk/TBS-T overnight at 4 °C, then rinsed in TBS-T. Membranes were then incubated for 30 min at RT in HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc, USA) diluted in 5% milk or BSA in TBS-T. Separated membranes containing WesternC marker were incubated in Streptavidin HRP-conjugated label (1:20,000) and secondary antibody only. Excess secondary antibody and Streptavidin-HRP was removed by washing in 4 changes of TBS-T. Cut membranes were pieced back together and incubated in Clarity ECL detection reagent (Bio-Rad, UK) for 5 min then imaged using the ChemidocMP imaging system (Bio-rad, UK). Protein bands were quantified by densitometry using Image Lab v4.1 software (Bio-Rad, UK). For Catalase and GP-1 Western blotting samples were prepared as described earlier, however proteins were electrophoresed through Bio-Rad Any kD mini-TGX PAGE gels alongside Kaleidoscope and Western C markers (Bio-Rad, UK), then transferred to PVDF using a Turbo Transfer blotter (2.5 mA, 25 V constant for 10 min). Membranes were blocked as earlier in 5% Milk/TBS-T then probed overnight with mouse anti-Catalase primary antibody (Sigma #C0979, diluted 1:1000 in 5% milk/TBS-T) at 4 °C then rinsed in TBS-T buffer. Membranes were then incubated for 30 min at RT in HRP-conjugated secondary antibody (Santa Cruz, Biotechnology, Inc, USA) diluted in 5% milk in TBS-T and imaged as described previously. Where necessary, membranes were stripped using Restore reagent (Thermofisher, UK) then reprobed for the housekeeping gene GAPDH (1:2000 Cat# MCA4740, Bio-Rad, UK) as a loading control.