Immobilon psq transfer pvdf membrane

To test the signaling pathways involved in the anti-proliferative effects of NDAT, gefitinib, and their combination, we applied western blot to quantify the protein expression levels in the total cell lysates of the primary cultures of human CRC cells (Colo_160224 cells); the cells were treated with NDAT, gefitinib, or in combination for 24 h. Protein samples were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel. The resolved proteins were transferred to Millipore Immobilon-PSQ Transfer PVDF membranes (Millipore, Billerica, MA, USA) by the Mini Trans-Blot Cell (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% skim milk in TBST, and incubated with primary antibodies against PD-L1 and GAPDH (GeneTex International Corp., Hsinchu City, Taiwan) at 4 °C overnight. HRP-conjugated secondary antibodies and the Immobilon TM Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore, Billerica, MA, USA) were used to detect the target antigen. Western blots were visualized and recorded with the BioSpectrum Imaging System (UVP, LLC, Upland, CA, USA). The densitometric analyses of western blots were analyzed with the ImageJ 1.5 software (NIH, Bethesda, MD, USA).

Nematodes were synchronized and treated for 1 day with or without 50‐mM metformin starting at L1/L2 or L4 larvae stage. After worms were homogenized in liquid nitrogen, the homogenate was lysed on ice for 60 min in lysis buffer (BioTeKe). Total proteins were loaded (40 μg per well) and separated on a 10% SDS polyacrylamide gel. Proteins were then transferred to Immobilon‐PSQ transfer PVDF membranes (Millipore, Bedford, MA). Primary antibodies against H3K4me3 (1:1,000 dilution; Abcam, ab8580), H3K9me3 (1:1,000 dilution; Abcam, ab8898), H3K27me3 (1:1,000 dilution; Abcam, ab272165), H3K36me3 (1:1,000 dilution; Abcam, ab9050), H3(1:2,000 dilution; Abcam, ab1791), p‐AMPKa (1:1,000 dilution, CST, 2535), p‐S6K (1:1,000 dilution, CST, 9205), S6K(1:1,000 dilution, CST, 2708), p‐mTOR (1:1,000 dilution, CST, 2971), mTOR (1:1,000 dilution, CST, 2972), and beta‐actin (1:1,000 dilution; Abcam, ab227387) used. The secondary antibody was a peroxidase‐coupled anti‐rabbit IgG (1:20,000 dilution; Abmart). The blots were developed using SuperSignal chemiluminescence substrate (Pierce). Band intensities were measured using ImageJ software.

Western blotting analyses were conducted followed routine protocol described previously (6 (link), 7 (link), 19 (link)–21 (link), 23 (link)). Consistent amount of protein samples were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). A 20 μg quantity of protein was loaded in each well with 5x sample buffer, and the protein samples were resolved with electrophoresis at 100 V for 2 h. The resolved proteins were transferred from the polyacrylamide gel to Millipore Immobilon-PSQ Transfer PVDF membranes (Millipore, Billerica, MA, USA) with the Mini Trans-Blot® Cell (Bio-Rad Laboratories). The membranes were blocked with a solution of 2% FBS in Tris-buffered saline and incubated with primary antibodies to pERK1/2 (GeneTex, Inc., Hsinchu, Taiwan), Cyclin D1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ERK1/2 (Santa Cruz Biotechnology), and α-Tubulin (Novus Biologicals, Littleton, CO, USA) at 4°C overnight and washed; the proteins were detected with HRP-conjugated secondary antibodies and Immobilon™ Western HRP Substrate Luminol Reagent (Millipore). Images of the western blots were visualized and recorded with an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The blots were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).