Ab227387

Nematodes were synchronized and treated for 1 day with or without 0.2 μM sanguinarine starting at L4 larvae stage. After worms were homogenized in liquid nitrogen, the homogenate was lysed on ice for 60 minutes in lysis buffer (BioTeKe). The lysates of total protein were loaded (40μg per well) and separated on a 10% SDS polyacrylamide gel. Proteins were then transferred to immobilon-PSQ transfer PVDF membrane (Millipore, Bedford, MA). Phosphorylated PMK-1 protein was detected using an anti-active p38 polyclonal antibody from rabbit (1:1000 dilution; Abcam, ab4822), anti-p38 antibody from rabbit (1:1000 dilution; Abcam, ab170099) and anti-beta actin antibody (1:1000 dilution; Abcam, ab227387). The secondary antibody was a peroxidase-coupled anti-rabbit IgG (1:20000 dilution; Abmart). Blots were developed using Super Signal chemiluminescence substrate (Pierce). Band intensities were measured using Image J software.

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.

KYSE-150 and KYSE-150R cells were collected and lysed using RIPA Lysis and Extraction Buffer (Thermo Scientific), and protein concentration was measured by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skim milk at room temperature for 2 h, then incubated with primary antibodies against E-cadherin (1:10000; ab40772, Abcam), vimentin (1:2000; ab92547, Abcam), snail (1:200; ab82846, Abcam), β-catenin (1:5000; ab227499, Abcam), Wnt3 (1:1000; ab32249, Abcam), β-actin (1:5000; ab227387, Abcam), histone H3 (1:1000; MA5-15150, Abcam) overnight at 4°C. The membrane was subsequently incubated with HRP-conjugated secondary antibodies (1:5000) at room temperature for 1.5 h. The proteins were visualized using enhanced chemiluminescence (Thermo Scientific), and the band intensity was determined by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Total proteins from cells and tissues were extracted, and the concentrations were determined using a bicinchoninic acid kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The extracted proteins were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis with the voltage increasing from 80 V to 120 V. Then the proteins were transferred onto polyvinylidene fluoride membranes using the semi-dry method at 80 mV for 30–45 min. The membranes were incubated with 5% bovine serum albumin at room temperature for 1 h, and then incubated with the primary antibodies against OPG (1:1000, ab2302), receptor activator of nuclear factor kappa B (RANK, 1:1000, ab32370), RANK ligand (RANKL, 1:5000, ab32064) and β-actin (1:5000, ab227387) (all purchased from Abcam) at 4 °C overnight. Afterwards, the membranes were washed with tris-buffered saline tween (TBST) (3 × 5 min), and incubated with the corresponding secondary antibody horseradish peroxidase-labeled immunoglobulin G (ab6747, 1:10000, Abcam) at room temperature for 1 h, After 3 times of TBST washes (5 min for each), the bands were visualized using chemiluminescence reagent on a Bio-Rad Gel Dol EZ imager (Bio-Rad Laboratories, California, USA). The target band was analyzed by calculating the gray value using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).