The cells were lysed and isolated in NETN lysis buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) supplemented with 1 mM PMSF (Beyotime, Haimen, China) [13 (link)]. Protein concentration was assessed using a Quick Start Bradford protein assay kit (Bio-Rad). Equal amount of total protein was boiled and separated by 10% SDS-PAGE and transferred onto the PVDF membrane. The blotted membrane incubated with diluted specific antibodies at 4°C overnight and goat anti-rabbit secondary antibody (1 : 10000, Jackson ImmunoResearch, USA) subsequently. The target bands were detected by ECL chromogenic substrate. STAT3 (#12640) and pSTAT3 (#9145) antibodies were purchased from Cell Signaling Technology Inc. PTPRK (ab185370) and β-actin (ab8227) antibodies were purchased from Abcam. Western blot quantification was performed using ImageJ software.
P stat3 9145
Manufactured by Cell Signaling Technology
Sourced in United States
P-STAT3 (#9145) is an antibody product from Cell Signaling Technology. It is designed to detect phosphorylated STAT3 protein.
Automatically generated - may contain errors
Lab products found in correlation
Stat3 12640, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab126605, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab246514, by Abcam (1 mentions)
Ab220161, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab5984, by Abcam (1 mentions)
Nupage system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Detroit 562, by American Type Culture Collection (1 mentions)
Mk2206, by Cell Signaling Technology (1 mentions)
Sch772984, by Cell Signaling Technology (1 mentions)
Mmp 2 40994, by Cell Signaling Technology (1 mentions)
15 protocols using p stat3 9145
1
Protein Extraction and Western Blotting
2
Immunoblotting of EMT and m6A Regulators
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Immunoblotting was carried out as previously described [44 (link)]. Antibodies against E-cadherin (3195, 1:1000), Vimentin (5741, 1:1000), p-STAT3 (9145, 1:1000), ZEB1 (70512, 1:1000) and β-actin (4970, 1:1000) were purchased from Cell Signaling Technology. Antibodies against FTO (ab126605, 1:1000), YTHDF1 (ab252563, 1:1000), YTHDF2 (ab246514, 1:1000), YTHDF3 (ab220161, 1:1000), TSG101 (ab125011, 1:1000), and CD81 (ab134045, 1:1000) were purchased from Abcam. Antibodies against IGF2BP1 (22803-1-AP, 1:1000), IGF2BP2 (11601-1-AP, 1:1000), IGF2BP3 (14642-1-AP, 1:1000) and GAPDH (60004-1-Ig, 1:2000) were purchased from Proteintech (China). Full and uncropped blots were uploaded as Supplemental Material.
3
Western Blot Analysis of Phosphorylated STAT3 in Heart Tissue
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Whole ventricle tissues or infarcted areas of heart tissues were homogenized in lysis buffer containing 25 mM HEPES (pH 7.5), 1% Triton X 100, 150 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The tissue extracts were resolved by the Nu-PAGE system (Thermofisher Scientific, Waltham, MA), and Western blot analysis performed as described previously [17 (link), 21 (link)]. Antibodies against tyrosine-phosphorylated STAT3 (p-STAT3; #9145, 500 ng/mL) and STAT3 (#12640, rabbit polyclonal, 23 ng/mL) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; MAB374, 6C5, 200 ng/mL) was purchased from Merck Millipore (Darmstadt, Germany). Antibody against IL-22R1 (ab5984, 1 μg/mL) was purchased from Abcam (Cambridge, United Kingdom). Antibody against IL-10RB (15102-1-AP, 450ng/mL) was purchased from Proteintech (Chicago, IL).
4
HNSCC Cell Lines and Inhibitors
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HNSCC cell lines Detroit 562, FaDu, and SCC-9 were obtained from ATCC (Manassas, VA USA).
The following inhibitors from Selleckchem (Houston, TX, USA) were used as follows: foretinib (0.5 µM), MK-2206 (1 µM), S3I-201 (100 µM), SCH772984 (1 µM), and trametinib (25 nM).
Antibodies for PD-L1 (13684), Erk1/2 (9107), P-Erk1/2 (4370), Vinculin (13901), Akt (4691), P-Akt (4060), Stat3 (9139), and P-Stat3 (9145) were obtained from Cell Signaling (Danvers, MA, USA). APC conjugated PD-L1 antibody (17-5983-41), and tubulin-antibody (MS-481-P) were purchased from ThermoFisher Scientific (Waltham, MA, USA), β-Actin antibody (A5316) from Merck, and PE conjugated Met antibody (FAB3582P) from R&D Systems (Minneapolis, MN, USA). All antibodies were applied as stated in the instructions of the manufacturers.
The following inhibitors from Selleckchem (Houston, TX, USA) were used as follows: foretinib (0.5 µM), MK-2206 (1 µM), S3I-201 (100 µM), SCH772984 (1 µM), and trametinib (25 nM).
Antibodies for PD-L1 (13684), Erk1/2 (9107), P-Erk1/2 (4370), Vinculin (13901), Akt (4691), P-Akt (4060), Stat3 (9139), and P-Stat3 (9145) were obtained from Cell Signaling (Danvers, MA, USA). APC conjugated PD-L1 antibody (17-5983-41), and tubulin-antibody (MS-481-P) were purchased from ThermoFisher Scientific (Waltham, MA, USA), β-Actin antibody (A5316) from Merck, and PE conjugated Met antibody (FAB3582P) from R&D Systems (Minneapolis, MN, USA). All antibodies were applied as stated in the instructions of the manufacturers.
5
Protein Expression Analysis of Key Signaling Pathways
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Western blot analysis was used to evaluate the expression of AKT2, HK2, MMP2, STAT3, and p-STAT3 in AGS and SGC-7901 cells, respectively. A total of 5 primary antibodies including AKT2#2964, HK2#2867, MMP2#40994, STAT3#12640, and p-STAT3#9145 were purchased from Cell Signal Technology, Inc. (USA). GAPDH antibody was purchased from abMart Company (Shanghai, China). First, the band (expression intensity) was checked by Pierce™ ECL substrate then measured by ChemiDoc™ Touch Imaging System (Bio-Rad, USA). The GAPDH expression was detected in a similar sample as a control. The dilution of the primary antibody was performed based on the manufacturer’s instructions.
6
Hepatocellular Carcinoma Cell Culture
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Human HCC cell SMMC‐7721 was purchased from Cell Bank of Xiangya Central Laboratory, Central South University. HepG2 was obtained from the Cell Bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). They were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37 °C in a humidified atmosphere with 5% CO2. Sorafenib tosylate, regorafenib (BAY 73‐4506), U0126, MK2206 2 HCl and S3I‐201 were purchased from Selleck (Selleck Chemicals, Shanghai, China). Recombinant human HGF protein was purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies against PARP (9532), Snail (3879), E‐cadherin (3195), vimentin (5741), α‐tubulin (2144), MET (8198), phosphorylated (P)‐MET (3077), ERK (4695), P‐ERK (4370), Akt (2920), P‐Akt (4060), STAT3 (12640) and P‐STAT3 (9145) were purchased from Cell Signaling Technology (Beverly, MA, USA). The primary antibody against GAPDH (AB22131) was obtained from Bioworld Technology, Inc. (St Louis Park, MN, USA).
7
Semi-Synthesis and Evaluation of M. gagei Derivatives
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MG was extracted from the heartwood of M. gagei, whereas MG ether derivatives were semi-synthesized according to the previous study [30 (link)]. Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), CDDP, MTT, and protease inhibitor were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA lysis buffer was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The protein assay reagents were purchased from Bio-Rad (Hercules, CA, USA). Human epidermal growth factor (EGF, 8916) and antibodies against phospho-EGFR (p-EGFR, 2234), total-EGFR (t-EGFR, 4267), phospho-STAT3 (p-STAT3, 9145), total-STAT3 (t-STAT3, 12640), phospho-Akt (p-Akt, 4060), total-Akt (t-Akt, 4691), phospho-Erk (p-Erk, 4377), total-Erk (t-Erk, 4695), caspase-3 (9662), PARP (9542), GAPDH (5174), and anti-rabbit IgG HRP-linked antibody (7074) were purchased from Cell Signaling Technology (Santa Cruz, CA, USA). The pan-caspase inhibitor Z-VAD(OMe)-FMK (ab120487) was purchased from Abcam (Cambridge, UK).
8
Luteolin and oxLDL Experiments
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Luteolin and oxLDL were obtained from Topscience Co., Ltd. (Shanghai, People’s Republic of China) and Yiyuan Biotechnology Co., Ltd., respectively (Guangzhou, People’s Republic of China). The Luteolin was dissolved in dimethyl sulfoxide (DMSO) and 1% sodium carboxyl methylcellulose (CMC-Na) for in vitro experiments and in vivo experiments. Moma-2 (MCA519) was purchased from Bio-Rad (Hercules, CA, USA). p-STAT3 (#9145) and STAT3 (#9139) were purchased from Cell Signaling (Danvers, MA, USA). β-actin (sc-47778) was obtained from Santa Cruz Technology (Delaware Avenue, CA, USA).
9
Comprehensive Antibody Validation Protocol
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STAT3 (sc-482), AR (sc-816), GAPDH (sc-47724), tubulin (sc-23948), ChgA (sc-1488), SYP (sc-17750) and ki67 (sc-23900) antibodies were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). H3K27me3 (#9733), H3K4me3 (#9751), EZH2 (#5246) and p-STAT3 (#9145) antibodies were from Cell Signaling Technology, Inc (Danvers, MA). The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX). RORα (GTX100029) antibody was from GeneTex (Irvine, CA).
10
Immunohistochemical Analysis of Neuronal Markers
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As previously described in Ref. [44] (link). Primary antibodies raised against CART H-003-63 and MCH H-070-47 (1:200; Phoenix Peptides, Buringlame, CA), CB1 sc20754, Y2 sc14736, and LepR sc8391 (1:100; Santa Cruz Biotechnology Inc., Dallas, TX), pSTAT3 9145 (1:100; Cell Signaling Technology, Beverly, MA), and CRE recombinase MMS-106P (1:200, Covance Inc, Emeryville, CA) were used. Secondary antibodies were used as appropriate and included donkey anti-rabbit immunoglobulin and donkey anti-goat immunoglobulin conjugated with Alexa Fluor 488 or 555 (1:400; Molecular Probes, Eugene, OR). Percentage of positive pixels and positive neurons were quantified using Scion software as previously described.
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