The suitably treated A546 and NCI-H460 cells were homogenized with RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Beyotime Biotechnology). The protein concentration of the lysates was measured using bicinchoninic acid assay (BCA). Equal amounts of protein per sample were separated by 10% SDS-PAGE and then electro-transferred to a PVDF membrane for 40 min at 4°C. After blocking with 5% skimmed milk/Tris-Tween buffer saline (TBST) for 1h at room temperature, the membranes were incubated overnight with the primary antibodies against ERK, p-ERK, MEK, p-MEK (1:1000, Cell Signaling Technology, CST), BAX, BCL-2 and β-actin (1:1000, Abcam, MA, USA). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG for 60 min. The positive bands were detected using an electrochemiluminescence (ECL) reagent.
P mek
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
P-MEK is a laboratory reagent that specifically detects phosphorylated MEK, a key signaling protein involved in various cellular processes. This product is intended for research use only and its performance characteristics have been validated by Cell Signaling Technology.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by Cell Signaling Technology (81 mentions)
P akt, by Cell Signaling Technology (40 mentions)
Gapdh, by Cell Signaling Technology (20 mentions)
β actin, by Cell Signaling Technology (19 mentions)
P erk1 2, by Cell Signaling Technology (19 mentions)
Erk1 2, by Cell Signaling Technology (14 mentions)
P jnk, by Cell Signaling Technology (13 mentions)
Pvdf membrane, by Merck Group (12 mentions)
P stat3, by Cell Signaling Technology (9 mentions)
C raf, by Cell Signaling Technology (9 mentions)
β actin, by Merck Group (9 mentions)
Cleaved caspase 3, by Cell Signaling Technology (9 mentions)
β actin, by Santa Cruz Biotechnology (9 mentions)
P raf, by Cell Signaling Technology (8 mentions)
P c raf, by Cell Signaling Technology (8 mentions)
Bcl 2, by Cell Signaling Technology (8 mentions)
Cleaved parp, by Cell Signaling Technology (7 mentions)
Snail, by Cell Signaling Technology (7 mentions)
P egfr, by Cell Signaling Technology (6 mentions)
Vimentin, by Cell Signaling Technology (6 mentions)
139 protocols using p mek
1
Western Blot Analysis of Signaling Pathways
2
Comprehensive Protein Analysis via Western Blot
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Western blot was conducted as previously described [23 (link)]. Primary antibodies against Cyclin-D, p-Rb, Rb, Capase 3, p-STAT3, STAT3, LMNB1, p-JAK, JAK, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, p-AKT, and AKT were purchased from Cell Signaling Technology. Antibodies against MVP and GAPDH were purchased from Santa Cruz Biotechnology. Quantification was performed with Image J.
3
Protein Expression Analysis in UVB-irradiated Skin
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Protein was extracted from the skin tissue of UVB-irradiated hairless mice. An equal concentration of protein samples (20 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. After transfer, the PVDF membranes were blocked for 1 h at room temperature with a 5% blocking solution (ATTO, Tokyo, Japan). After blocking, they were incubated at 4 °C overnight with indicated primary antibodies (MMP-1 (#54376), MMP-9 (#13667), pERK (#9101), ERK (#9102), pMEK (#9154), MEK (#9126), pp38 (#9215), p38 (#9212), pJNK (#9251), JNK (#9252), and β-actin (#4970) were purchased from Cell signaling, diluted (1:1000), washed three times for 10 min each in Tris-buffered saline (TBS), and then incubated for 2 h at room temperature with the anti-rabbit secondary antibody (#7074, Cell signaling). The proteins were developed by an enhanced chemiluminescence (ECL) solution and detected with a LAS-4000 mini luminescent image analyzer (GE Heathcare, UK).
4
Protein Expression Analysis in Transfected Cells
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Cells transfected with siRNA, plasmid, lentivirus, miR‐6887‐3p mimics or miR‐6887‐3p inhibitor were harvested in sample buffer supplemented with Protease/Phosphatase Inhibitor Cocktail (#5872, Cell Signaling Technology, Shanghai, P.R. China). Protein levels were detected and calculated using the bicinchoninic acid (BCA) assay (#AR0197A, BOSTER, Shanghai, P.R. China) according to the manufacturer's instructions. Equal amounts (20 µg) of protein were added into sodium dodecyl sulfate (SDS) polyacrylamide gel, and the gel was transferred to polyvinylidene difluoride membranes (#10600023, GE, Shanghai, P.R. China). The membrane was incubated with primary antibodies overnight after blocking by milk. Then, the membrane was incubated with second antibody to be photographed. All used antibodies were Mex3a (1:1000 dilution; #ab79046, Abcam), RAP1GAP (1:5000 dilution; #ab32373, Abcam), MEK (1:500 dilution; #8727, Cell Signaling Technology, Shanghai, P.R. China), p‐MEK (1:500 dilution #9154, Cell Signaling Technology, Shanghai, P.R. China), ERK (1:1000 dilution; #4695, Cell Signaling Technology, Shanghai, P.R. China), p‐ERK (1:1000 dilution; #4370, Cell Signaling Technology, Shanghai, P.R. China), HIF‐1α (1:1000 dilution; #ab51608, Abcam, Cambridge, UK) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (1:5000 dilution; #2118, Cell Signaling Technology, Shanghai, P.R. China).
5
Cell Signaling Pathway Analysis
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Fluorouracil, selumetinib, BIO and NAC were purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO or diluted water. DCF-DA was purchased from the Life Technology (Invitrogen, Carlsbad, California, USA) and dissolved in DMSO. Melatonin was from Sigma Aldrich (Sigma-Aldrich, St. Louis, USA). Antibodies include PARP, pAkt, Akt, pErk, Erk, GSK3β, pGSK3β, MEK, pMEK, β-Actin and GAPDH (Cell Signaling Technology, Beverly, MA, USA) as well as Ki-67 (Abcam, Cambridge, Massachusetts, USA).
6
Western Blot Analysis of Signaling Proteins
7
Western Blotting of Signaling Proteins
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Cells were added to non-ionic detergent-containing buffers (RIPA lysis buffers, Beyotime Biotechnology, Shanghai, China) 24 h after SCC-9 cell line transfection, and the protein concentration was determined with bicinchoninic acid (BCA, Thermo Fisher Scientific Inc., Waltham, MA, USA).Protein extractswere heated at 100°Cfor 5 min (20 μL samples). A total of 10% polyacrylamide gel was used to separate protein via electrophoresis at 48 V for 3.5 h with a Polyvinylidene Fluoride (PVDF) transmembrane. Samples were then blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and rinsed with tris-buffered saline-tween (TBST) once. The primary antibody, 5% BSA with the addition of FAK, pFAK, ERK, pERK, MEK, pMEK, and β-actin (Cell Signaling Technology, Beverly, MA, USA), was added to the sample for incubation for 1 h at room temperature, followed by TBST rinsing once. The secondary antibody (goat anti-mouse and goat anti-rabbit, ABCAm Inc., Cambridge, MA, USA) was added, kept at room temperature for 1 h, rinsed once with TBST, and enhanced chemiluminescence (ECL) was used for development. Relative optical density (OD) analysis was performed on all the bands of western blotting. The relative expression of the target protein was calculated from the ratio of integrated OD of the target band and that of the corresponding internal reference.
8
Signaling Pathway Protein Analysis
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SDS-PAGE and immunoblotting were performed according to a standard procedure. ECM1, HER3, phosphorylated ERK (p-ERK), ERK, actin, MUC1 and galectin-3 antibodies were obtained from Santa Cruz Biotechnology. c-Raf, p-c-Raf, MEK, p-MEK, Akt, p-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Epidermal growth factor receptor (EGFR) antibody was obtained from Abcam (Cambridge, UK). HER2 antibody was obtained from Thermo Scientific (Waltham, MA, USA).
9
Immunohistochemical Analysis of p-MEK and B7-H3
10
Western Blot and Immunohistochemistry Protocol
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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)].
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