Hrp conjugated anti secondary igg antibodies

Sample preparation was conducted as per methods described previously [36 (link)]. Protein samples were separated using 4–20% Mini-PROTEIN TGX^TM Precast Gels and transferred onto nitrocellulose membranes (0.45 μm) using the Trans-Blot Turbo Blotting System (Bio-Rad, Hercules, CA, USA). Blocking was performed using the Everyblot Blocking buffer (Bio-Rad). Membranes were then incubated with primary antibodies, containing recombinant anti-TMEM16A antibody [SP31] (ab64085) (Abcam, Cambridge, UK), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody #9101 (cell signaling, Danvers, MA, USA), p44/42 MAPK (Erk1/2) antibody #9102 (cell signaling), phospho-EGF Receptor (Tyr1068) (D7A5) XP^® rabbit mAb #3777 (cell signaling), anti-EGFR antibody (A-10) (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (Santa Cruz Biotechnology) overnight. The membranes were then incubated with HRP-conjugated anti-secondary IgG antibodies (Enzo Life Science, Farmingdale, NY, USA) for 1 h at RT. Finally, visualization was performed using the Clarity^TM Western ECL substrate with ChemiDoc (Bio-Rad).

Preparation of the protein sample was carried out as described previously [45 (link)]. The samples were separated by 4–12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Republic of Korea) and then transferred onto a polyvinylidene Fluoride membrane (Millipore, Billerica, MA, USA). Blocking of the membrane was performed with 5% non-fat skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membrane was incubated overnight at 4 °C with corresponding primary antibodies, including anti-ANO1 (ab64085; Abcam, Cambridge, UK), anti-β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-cleaved PARP (551,025; BD Biosciences, Franklin Lakes, NJ, USA) antibodies and then was washed three times with TBST followed by 1 h of incubation with HRP-conjugated anti-secondary IgG antibodies (Enzo Life Science, Farmingdale, NY, USA) at room temperature. Visualization was performed using the SuperSignal™ Western Blot Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Sample preparation was performed as described previously [17 (link)]. The protein samples were separated by 4–12% Tris-Glycine-PAG Pre-Cast Gel (KOMA BIOTECH) and transferred to polyvinylidene Fluoride (PVDF) membranes. Blocking was done using 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. Membranes were then incubated with corresponding primary antibodies, including anti-ANO1 (Abcam), anti-β-actin (Santa Cruz Biotechnology), and anti-cleaved PARP (BD Biosciences) antibodies, followed by incubation of HRP-conjugated anti-secondary IgG antibodies (Enzo life science) for 1 h. Finally, visualization was done with the ECL Plus Western Blotting System (GE Healthcare).