Protein extraction and Western blot analysis were performed as described before.17 (link) Antibodies against specific proteins, including VEGF (Abcam, ab214424), PIAS3 (Cell Signaling Technology, 9042), p-STAT3 (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 12640), CD63 (Abcam, ab134045) and TSG101 (Abcam, ab125011), were used.
Pias3
Manufactured by Cell Signaling Technology
Sourced in United States
PIAS3 is a protein product offered by Cell Signaling Technology. It functions as a SUMO E3 ligase, which catalyzes the conjugation of SUMO to target proteins.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (5 mentions)
P stat3, by Cell Signaling Technology (4 mentions)
Stat3, by Cell Signaling Technology (3 mentions)
Total stat3, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ab134045, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab214424, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Complete mini protease inhibitor tablet, by Roche (1 mentions)
Stat3 c 20, by Santa Cruz Biotechnology (1 mentions)
Recombinant lif, by Merck Group (1 mentions)
P stat3 y705, by Cell Signaling Technology (1 mentions)
Socs3, by Abcam (1 mentions)
Phorbol myristate acetate (pma), by LC Laboratories (1 mentions)
10 protocols using pias3
1
Protein Extraction and Western Blot Analysis
2
Western Blotting Analysis of Protein Signaling
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Western blotting was performed as previously described44 (link). Protein extracts of cells and tissues were prepared using RIPA lysis buffer (Sigma-Aldrich), and the total protein content was quantified by the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The antibodies included PIAS3, p27, p-STAT3 and total-STAT3 (1:1000 dilution, Cell Signaling Technology, Boston, MA, USA). The primary antibodies were incubated overnight at 4 °C followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The protein expression was normalized by blotting the same membranes with an antibody against GAPDH (1:10000 dilution, KangCheng Bio-tech, Shanghai, China).
3
Quantitative Western Blot Analysis of Lung Cancer Biomarkers
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Cell line lysates were prepared in RIPA buffer as previously described 15 (link),16 (link). Protein pellets of surgically resected human squamous cell lung cancer specimens were obtained from the Lung Cancer Biospecimen Resource Network (LCBRN, University of Virginia). The dried protein precipitate pellet was suspended in buffer containing 5% SDS, 1% Triton, 50 mmol/L Tris, and 150 mmol/L sodium chloride containing a Complete Mini Protease Inhibitor Tablet (Roche, Indianapolis, IA) and sonicated for 7–10 sec on ice. Protein was quantified by the Pierce BCA protein assay kit (ThermoFisher Scientific). All protein samples were electrophoretically resolved through 4–20% gradient gels (Bio-Rad, Hercules, CA). Antibodies used were PIAS3 (#4164; Cell Signaling Technology, Danvers, MA), STAT3 (#sc-7179, Santa Cruz Technology, Santa Cruz, CA), and β-Actin (#A-5441, Sigma-Aldrich, St. Louis, MO). The working antibody dilutions were: PIAS3 (1:1000), STAT3 (1:2000), β-actin (1:50,000). Secondary antibodies were used at 1:10,000.
4
Characterization of STAT3 Signaling
5
Comprehensive Western Blot and IF Analysis
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Western blots were performed using 10% standard gels from Bio‐Rad (Hercules, CA, USA). Proteins were isolated from cells as described (Mukherjee et al., 2016 ). The following antibodies were applied for western blot analysis or IF analysis: TRIM8 (NBP1‐89776; NOVUS), PIAS3 (9042; Cell Signaling, Danvers, MA, USA), STAT3 and phosphor‐STAT3 (PSTAT3‐Y705) (Cell Signaling), GFP (TA180076; Origene), β‐actin (A5441; Sigma, St. Louis, MO, USA), GAPDH (GTX627408; GeneTex, Irvine, CA, USA), GFAP (3670; Cell Signaling), NESTIN (ab6320; Abcam, Eugene, OR, US), SOX2 (2748; Cell Signaling), CD133 (W6B3C1; Miltenyi), and c‐MYC (ab32072, Abcam).
6
Western Blotting and qPCR Analysis of Signaling Proteins
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Western blotting was performed as described previously using specific antibodies against RORα, actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), pSTAT3, STAT3, JAK1, JAK2, SRC, PIAS3, and SHP-1 (Cell Signaling Technology, Beverly, MA, USA)32 (link). Quantitative real-time PCR (qPCR) was performed using an ABI StepOnePlusTM Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using specific primers (Supplementary Table S1 ). Relative mRNA levels of target genes were estimated using the equation 2−ΔCt (ΔCt = Ct of target gene minus Ct of β-actin or 18 S rRNA) and are presented relative to the level of the control group, which was designated as 1. The detailed method for qPCR is described in Han et al.32 (link).
7
Protein Lysate Analysis by Western Blot
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Protein lysates were prepared and analyzed on 4–20% Criterion gels (Bio‐Rad, Hercules, CA, USA) as described previously (Unkila et al., 2001 ). Primary antibodies used were as follows: PIAS3 (Cell Signaling Technology, Danvers, MA, USA, #4164), TP53 (Santa Cruz Biotechnology, Dallas, TX, USA, sc‐71817), survivin (Cell Signaling Technology #2803), and beta‐actin (Sigma, St. Louis, MO, USA, A‐5441).
8
Bufalin Signaling Pathway Analysis
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Bufalin was purchased from Sigma (Cat# B0261) (St. Louis, MO, USA). Antibodies to PIAS3 (Cat# 9042, RRID: AB_2797692), STAT3 (Cat# 12640, RRID: AB_2629499), p-STAT3 (Cat# 9145, RRID: AB_2491009), and β-actin (Cat# 3700, RRID: AB_2242334) were purchased from Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; Cat# AP187P), and HRP-conjugated goat anti-mouse IgG (Cat# AP181P) were purchased from Sigma (St. Louis, MO, USA).
9
Profiling Synovial Fibroblast Signaling in Psoriatic Arthritis
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Protein isolation from Psoriatic Arthritis synovial fibroblasts (PsAFLS) and synovial explants is described in online supplementary file 1. Protein (20–50 µg) was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking, 10% resolving), gels were then transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK) prior to 1 h blocking in wash buffer containing 5% non-fat milk. Membranes were incubated with rabbit polyclonal anti-pSTAT3 (Cell-Signaling Technology, UK), total-signal transducer and activator of transcription (tSTAT)3, pSTAT1, tSTAT1, pSTAT2, suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3; Cell Signaling Technology) and nuclear factor kappa B cells (NFκBp65) (Millipore, California, USA) diluted in 5% non-fat milk containing 0.1% Tween 20 at 4°C overnight. β-Actin (Sigma-Aldrich) was used as a loading control. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 3 h at RT. Signal was detected using SuperSignal West-Pico Chemiluminescent Substrate (Amersham Biosciences, UK) and quantified using EDAS-120 system (Kodak, Rochester, New York, USA).
10
Comprehensive Protein Immunodetection Protocol
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Anti-caveolin-1 (N-20) and DUSP5 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- cleaved caspase-9, cleaved PARP, phospho-Akt, total Akt, phospho-BRCA1, phospho-DNAPK, total BRCA1, total DNAPK, phospho-JAK2, total JAK2, phospho-STAT3, total STAT3, PIAS3, Src (Y416), Src (Y527), total Src, SOCS2, phospho-JNK, total JNK, phospho-p38, total p38, phospho-ERK1/2, total ERK1/2, alpha tubulin, phospho-H2.AX, beta actin and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). Gemcitabine and 5-fluorouracil (5-FU) was purchased from Sigma-Aldrich (St. Louis, MO) and Santa Cruz Biotechnology (Dallas, TX) respectively. For in vitro studies, Gemcitabine and 5-FU were dissolved in DMSO. MIAPaCa-2, BxPC3, Panc-1, AsPC1, Capan-1, Capan-2, HPAFII and HEK-293 were obtained from and authenticated (via short tandem repeat profiling) by the American Type Culture Collection (Manassas, VA), and grown according to ATCC recommendations. HPDE cells were kindly provided by Dr Diane Simeone (University of Michigan). Cells used for this study were cryopreserved within 6 months of authentication. SW-48 and DLD-1 isogenic cell line pairs were obtained from Horizon Discovery (Cambridge, UK). Cells were passaged for no longer than 3 months and grown in a 37 °C incubator with 5% CO2.
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