Lung nodules were homogenized, lysed using RIPA (Santa Cruz, TX), and 10% SDS/PAGE was run after protein quantification (Bio‐Rad, Hercules, CA). Primary antibodies (1:1000): PD‐L1, p‐Stat3‐705, Stat‐3, phospho‐p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, cleaved caspase‐3 (Asp175), and caspase‐3 (Cell Signaling, MA) were used. Autoradiography detection and quantification were performed using Image J.
P stat3 705
Manufactured by Cell Signaling Technology
Sourced in United States
P-STAT3(705) is a primary antibody that targets phosphorylated STAT3 at tyrosine 705. It is used to detect the activated form of the STAT3 transcription factor in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
P stat3 727, by Cell Signaling Technology (3 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Rnaseout recombinant rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Odyssey xf imaging system, by LI COR (1 mentions)
Mab338, by Merck Group (1 mentions)
Image studio ver 5, by LI COR (1 mentions)
Mouse anti e cadherin, by Cell Signaling Technology (1 mentions)
8 protocols using p stat3 705
1
Lung Nodule Protein Profiling
2
Comprehensive Protein and Gene Expression Analysis
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RT-qPCR and Western blotting were performed, as we did previously (Jia et al., 2019 (link)). Primers from ThermoFisher Scientific included Mouse CNTF (Mm00446373-ml), LIF (Mm00434762_g1), IL-6 (Mm0044619_ml), TNF (Mm00443258_ml) and GAPDH (Mm99999915-gl). Data analysis was performed with ΔΔCt method and GAPDH was used as an endogenous loading control. The antibodies used in Western blots included CNTF antibody (MAB338, EMD Millipore, RRID: AB_2083064 ), phospho-STAT3-Tyr705 (pSTAT3705, #9131, Cell signaling, RRID: AB_331586 ), STAT3 (#9132, Cell Signaling, RRID: AB_823645 ), and β-actin (Cat# 4967, Cell Signaling, RRID: AB_330288 ). For fluorescence western blot, donkey anti-rabbit IRDye 800CW (926–32213, LI-COR) and anti-mouse IRDye 680RD (926–68072, LI-COR) secondary antibodies were used. For chemiluminescence western blot, HRP-conjugated secondary antibodies were used. Images were taken by Odyssey XF Imaging System (LI-COR) and quantified using Image Studio Ver 5.2 (LI-COR). Donkey anti-goat IgG-conjugated to Alex Fluor-488 was used for immunostaining. The levels of plasma corticosterone were measured using ELISA kit (ab108821, Abcam).
3
Western Blot Analysis of EMT and Apoptosis
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Cells were harvested, washed three times with PBS, and then lysed by using RIPA buffer. Protein concentrations were determined using BCA Protein Assay Reagent (Pierce Chemical, Rockford, IL). Equal amounts of total protein (50 μg per lysate) were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes at 250 V for 2 h. Membranes were blocked for 1 h with 5% (w/v) nonfat milk powder in Tris-buffered saline/0.1% Tween 20 and then incubated with primary antibodies: mouse anti-E-cadherin, N-cadherin, vimentin, TWIST STAT3, p-STAT3(705), BAX, AKT, p-AKT, BCL-2, cleaved caspase 3, and β-actin (Cell Signaling Technology, Beverly, MA) at 4°C overnight. The membranes were washed and incubated with a Dylight 800-conjugated goat anti-mouse secondary antibody (EarthOx, San Francisco, CA). Membranes were washed again, and the immunoblotted proteins were detected using an Odyssey Western Blotting Detection System (Gene, Hong Kong, China).
4
Inflammatory Signaling Pathway Analysis
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CM was obtained from Hsinhai Biotechnology (Taichung, Taiwan). Dimethyl sulfoxide (DMSO), dexamethasone (Dexa), paraformaldehyde, bovine serum albumin (BSA), LPS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 4′,6-diamidino-2-phenylindole (DAPI) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM/F12 medium, RPMI1640 medium, penicillin, streptomycin, and fetal bovine serum (FBS), LipofectamineTM 3000, TAK-242 (CLI-095), TRIzol™ Reagent, SuperScript™ II Reverse Transcriptase, RNaseOUT™ Recombinant RNase Inhibitor, Hoechst 33258, SYBR green, Alexa Fluor 488, and Alexa Fluor 594 were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies of PI3K, p-PI3K, Akt, p-Akt, IKK, p-IKK, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, c-Fos, Nrf2, p-Nrf2, SOD1, SOD2, CAT, HO-1, STAT1, p-STAT1, STAT3, p-STAT3(727), STAT3, p-STAT3(705), JAK1, p-JAK1, JAK2, p-JAK2, PCNA, and β-actin were obtained from Cell Signaling (Beverly, MA, USA).
5
Immunohistochemistry of Retina and Optic Nerve
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Mice were given a lethal dose of anesthesia (ketamine and xylazine) and then perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA) (Sigma-Aldrich). Retinas and optic nerves were dissected, fixed in 4% PFA overnight, and then dehydrated in 30% sucrose for 6 hours. The resulting retinas and optic nerves were embedded into optimal cutting temperature compound (Sakura) at −80°C and then cryosectioned at −20°C (25 μm for retinas and 8 μm for optic nerves). These sections were blocked with 0.1% Triton X-100 in 4% normal goat serum for 30 min and then incubated overnight with the primary antibodies, including Tuj 1 (neuron-specific class III beta-tubulin) (BioLegend, catalog no. 801202; mouse monoclonal antibody), p-STAT3 705 (Cell Signaling Technologies, catalog no. 9145; rabbit monoclonal antibody), and Alexa Fluor 488 (Invitrogen, catalog no. A-11094; rabbit polyclonal antibody). The sections were washed with PBS (three times) and then incubated with suitable secondary antibodies (Invitrogen) for 2 hours. After wash with PBS (three times), the sections were mounted on coverslips for imaging.
6
Western Blot for Protein Expression
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Cells or tumor tissues were collected and suspended in RIPA lysis buffer (Biomiga, Inc.) containing a cocktail of proteinase inhibitors (Roche). The protein concentration was quantified using the bicinchoninic acid (BCA) assay kit (Thermo scientific, Inc.) to ensure that equal amounts of protein from different subpopulations were loaded into the gel. The proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against murine GZMM (P-15, Santa Cruz Biotechnology, Inc.); E-cadherin, N-cadherin, vimentin, AKT, p-AKT, ERK1/2, p-ERK1/2, STAT3, p-STAT3705, p-STAT3727 (Cell Signaling Technology), a-tubulin (ZSGB-BIO), and β-actin (ZSGB-BIO) were used at 1:1000 dilution and incubated with the membranes at 4°C overnight. The reaction was revealed by horseradish peroxidase (HRP)-coupled secondary reagents (Pierce, Rockford, IL, USA), developed by enhanced chemiluminescence (ECL) (Applygen Technologies Inc.) according to the manufacturer's instructions, and subjected to exposure using LAS4010 (General Electric Company).
7
Thoracic Aorta Homogenization and Protein Analysis
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Thoracic aorta was grinded using homogenizer. The aortic homogenation and cultured VSMCs were lysed with 0.25 M Tris (pH 6.8), 1.5% SDS, and 1% mercaptoethanol on ice. The lysis samples were subjected to electrophoretic separation with 10% SDS polyacrylamide gel electrophoresis and transferred onto NC membrane (Millipore, Schwalbach, Germany) as described earlier [29 (link)]. Membranes were pre-incubated with 5% dry-milk powder incubated 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibody Y1R (1:500, Abcam), Y5R (1:500, Abcam), STAT3 (1:500, Cell Signaling), P-STAT3 (705) (1:500, Cell Signaling), P-STAT3(727) (1:500, Cell Signaling), c-Fos (1:500, PTG), goat polyclonal antibody NPY2R (1:500, Sigma), rabbit polyclonel antibody PCNA(1:1000, PTG), mouse polyclonal antibody GAPDH (1:1000, PTG), respectively. After incubation with alkaline phosphatase-conjugated secondary antibodies (Jackson Immunoresearch), the signals were visualized by nitroblue tetrazolium—bromochloroindolyl phosphate (Bio Basic, Inc.) and quantified with Quantity One software (Bio-Rad).
8
Anti-inflammatory mechanisms of PIPO
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PIPO was a gift from the Wide Pharmaceutical Co., Ltd. (Taichung, Taiwan). LPS (lipopolysaccharides from Escherichia coli O111:B4), 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), Griess reagent (1% sulfanilamide, 0.1% N‐1‐naphthylenediamine dihydrochloride, and 2.5% phosphoric acid), cell lysis buffer, and dimethyl sulfoxide were obtained from Sigma‐Aldrich (St. Louis, Missouri). Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Life/Gibco (Grand Island, New York). Antibodies against iNOS, COX‐2, NF‐κB/p65, NF‐κB/p50, Nrf2, HO‐1, p‐STAT1 (705), STAT1, p‐STAT3 (727), and p‐STAT3 (705), and STAT3 were obtained from Cell Signaling (Beverly, Massachusetts). TRIzol reagent, SuperScript II Reverse Transcriptase, RNaseOUT Recombinant RNase Inhibitor, Hoechst 33258, and Alexa Fluor 488 or 568 were obtained from Invitrogen (Carlsbad, California). Prostaglandin E2 ELISA Kit was from Cayman (Ann Arbor, Michigan).
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