Erk1 2

Cells were lysed on ice in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Beyotime, Haimen, China). Total and nuclear proteins were extracted according to the manufacturer’s instructions (Beyotime, Haimen, China). Protein samples (50 μg) were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked in TBST containing 5% fat-free milk at room temperature for 1 h. After washing with TBST, the membranes were probed with primary antibodies against C/EBPα, FABP4, PPARγ, RhoA, ROCK1, ROCK2, ERK1/2, β-actin, PCNA (all Proteintech, Wuhan, China), and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed in TBST and incubated with secondary antibody (Proteintech, Wuhan, China). The signals were developed by ECL reagents (Beyotime, Haimen, China).

Western blot was performed according to the previous report 8 (link). Briefly, total proteins were separated by 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% skim milk, the PVDF membrane was incubated with primary antibodies and HRP-conjugated secondary antibodies. The primary antibodies are as follows: Wnt5a (1:1000, Abcam), p-ERK1/2 (1:1000, CST), ERK1/2 (1:1000, Protein Tech), p-CaMKII (1:1000, CST), CaMKII (1:1000, Protein Tech), α-Tubulin (1:5000, Protein Tech), GAPDH (1:5000, Protein Tech).

Erlotinib-resistant HCC827/ER cells were established as previously described [34 (link)] and cultured in a RPMI-1640 medium (Hyclone, Beijing, China, SH30809.01) supplemented with 10% FBS (R&S, Australia, 009106), penicillin and streptomycin (Gibco, New York, NY, USA, 15140122), and 5 μM Erlotinib (Selleck, Shanghai, China, s1023) at 37 °C and 5% CO₂. The parental HCC827 cell line was purchased from ATCC. The following antibodies were used: p-AKT^Ser473 (Cell Signaling Technology, Danvers, MA, USA, 4060), AKT (Abcam, Shanghai, China, ab79360), E-cadherin (Proteintech, Wuhan, China, 20874-1-AP), N-cadherin (ZEN Bio, Chengdu, China, 382812), vimentin (ZEN Bio, Chengdu, China, R22775), snail (Cell Signaling Technology, 3879), laminA/C (Santa Cruz, Starr County, TX, USA, sc-376248), laminB1 (Cell Signaling Technology, 12586), ZEB1 (Cell Signaling Technology, 3396), p-FGFR (Abcam, Shanghai, China, ab192589), p-ERK1/2 (Cell Signaling Technology, 4370), ERK1/2 (Proteintech, Wuhan, China, 67170-1-Ig), p-c-fos (Santa Cruz, TX, USA, sc-81485), c-fos (ZEN Bio, Chengdu, China, 340249), p-EGFR^Y845 (Cell Signaling Technology, 6963), and GAPDH (SAB, MD, USA, 48142).

Western blotting was performed as described previously [20 (link)]. Samples of equivalent total protein (20 μg) were loaded. Primary antibodies against CD63, CD9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-MYC, p-EGFR, p-MEK, p-ERK(Cell Signaling Technology, Danvers, MA, USA), GAPDH, DNAJB11, HSPA5, ATF6, IRE1, XBP1, PERK, ATF4, EGFR, Raf-1, MEK, and ERK1/2 (ProteinTech Group, Rosemont, IL, USA) were used. Immunohistochemical (IHC) assay was performed following a previously described procedure [21 (link)].

Cell samples were lysed with 2×SDS lysis buffer (Beyotime Biotechnology, Shanghai, China). Tissues were grinded in liquid nitrogen and treated with RIPA (Beyotime Biotechnology, Shanghai, China). Quantification of proteins was performed using BCA (bicinchoninic acid assay). Protein samples were isolated in SDS-PAGE gel by electrophoresis and transferred to a PVDF membrane. GAPDH mouse (proteintech, Wuhan, China), p-ERK1/2 (Santa Cruz, United States), ERK1/2 (proteintech, Wuhan, China), p-JNK (proteintech, Wuhan, China), JNK (proteintech, Wuhan, China), KRAS (proteintech, Wuhan, China), anti-mouse IgG (H + L) (CST, United States), and anti-rabbit IgG (H + L) (CST, United States) were used to incubate the PVDF membrane according to the detecting proteins. Results were read via chemiluminescence (Tanon 5200 multi, Shanghai, China) using an ECL kit (Beyotime Biotechnology, Shanghai, China).

Cells were collected 48 h after transfection, and cell lysates were prepared using ice-cold Tris buffer (20 mmol/l Tris; pH 7.5) containing 137 mmol/l NaCl, 2 mmol/l EDTA, 1% Triton X, 10% glycerol, 50 mmol/l NaF, 1 mmol/l DTT, PMSF, and a protein phosphatases inhibitor (Applygen Tech., Beijing, China). For extracellular signal-regulated kinase (ERK) signaling analysis, cells were starved with serum-free medium for 24 h after transfection. These cells were then stimulated with a medium containing 10% serum for 45 min before collection. Western blot was performed as described previously [6 (link)]. Primary antibodies were as follows: TMEM176A (Sigma, St. Louis, MO), cleaved caspase-3 (Cell Signaling Technology, Danfoss, MA, USA), MMP2 (Bioworld Tech., MN, USA), MMP9 (Bioworld Tech., MN, USA), ERK1/2 (Protein Tech Group, Chicago, IL, USA), p-ERK1/2 (Cell Signaling Technology, Danfoss, MA, USA), SAR1A (Protein Tech Group, Chicago, IL, USA), and β-actin (Beyotime Biotech, Nanjing, China).

Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100 °C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4 °C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed five times for 30 min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 (1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII (1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).