Anti erk1 2

A subconfluent monolayer of cells was serum-deprived for 4 h in DMEM and then stimulated with 10 mM CaCl₂. Cells were harvested with Cell Lysis Buffer® (Cell Signaling, Beverly, MA, USA) according to the manufacturer's instructions. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). Membranes were blocked for 1 h in 5% (w/v) non-fat dried milk in phosphate-buffered saline with 0.1% (v/v) Tween 20 and incubated with anti-phosphorylated p38^{Thr180/Tyr182}, anti-p38, anti-ERK1/2, or anti-phosphorylated ERK1/2^{Thr202/Tyr204} (all Cell Signaling) at a 1:1000 dilution at room temperature for 1 h. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) at a 1:2000 dilution at room temperature for 1 h. Proteins were detected using ECL Prime Western blotting detection reagents (GE Healthcare, Madison, WI, USA) according to the manufacturer's instructions and visualized using Molecular Imager® ChemiDoc™ XRS plus (Bio-Rad).

B16-F10 cells (2 × 10⁶ cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).

The following antibodies were used according to the protocols supplied by the manufacturers: anti-pERK1/2 (#9101, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (#4695, Cell Signaling Technology), anti-GAPDH (ENM0040, Elabscience Biotechnology Inc., Houston, TX, USA), anti-pEGFR (12A3) (sc-57542, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-EGFR (C-2) (sc-377229, Santa Cruz Biotechnology Inc.), anti-pAKT (#9271, Cell Signaling Technology), anti-AKT (#9272, Cell Signaling Technology), anti-Cyclin D1 (sc-753, Santa Cruz Biotechnology Inc.), anti-p21 (sc-397, Santa Cruz Biotechnology Inc.), and anti-beta Tubulin (Sigma), anti-alpha actin (Sigma). All the secondary antibodies (HRP-conjugated anti-rabbit and anti-mouse) were purchased from Santa Cruz Biotechnology Inc.
Drug formulations: gefitinib (ZD1839) and MG132 (S2619) were purchased from Selleckchem; AZD9291 was obtained from AstraZeneca; BAPTA_AM (HB0981) was from HelloBio.
Recombinant Human EGF (AF-100-15) was from PeproTech (London, UK).

Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).

For the Western blot analysis, the liver tissues or primary hepatocytes were lysed in 1× radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktails (Roche Diagnostic GmbH, Mannheim, Germany). Protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). The cell lysate was loaded onto a 10% Sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis and then electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated with the indicated primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence solution (NEN Life Science, Boston, MA, USA). The following antibodies were used for the experiments: Anti-GNMT (catalog number: 14-1, YMAC Bio Tech, Taipei, Taiwan), anti-Akt (catalog number: #4685), anti-phospho-Akt T308 (catalog number: #13038), anti-phosphoAkt S473 (catalog number: #9271), anti-ERK1/2 (catalog number: #4695), anti-phosphoERK1/2 (catalog number: #4376) (Cell Signaling Technology, Danvers, MA, USA), anti-Angptl8 (catalog number: TA326696) (Origene, Rockville, MD, USA), and anti-Actin (Sigma-Aldrich) antibodies.