To assess the levels of protein expression using western blotting, cells or tissue were lysed in RIPA buffer with protease and phosphatase inhibitors. Western blotting was performed as described.8 (link) The phosphorylated protein kinase B (pAKT), AKT, p-p44/42 mitogen-activated protein kinase (MAPK) (phosphorylated extracellular-signal-regulated kinase 1/2), p44/42 MAPK (extracellular-signal-regulated kinase 1/2), phosphorylated signal transducer and activator of transcription (pSTAT)5, STAT5, STAT5A, STAT5B, pSTAT3, STAT3, BCL-2, cleaved caspase 3 (CC3), Lamin B1, and X-linked inhibitor of apoptosis protein antibodies were purchased from Cell Signaling (distributed through New England Biolabs GmbH). β-Actin and β-Tubulin antibodies were purchased from Santa Cruz Biotechnology, α-Tubulin antibody from Sigma-Aldrich. Phosphorylated AXL (pAXL) antibody (Y779), which detects human and mouse AXL when phosphorylated at Y779, a well conserved motif in humans (according to phosphosite.org), was purchased from R&D Systems. The AXL antibody was a gift from Björn Dahlbäck, Malmö, Sweden. For detailed information on the antibodies please refer to Supplemental Table 2 (http://links.lww.com/HS/A188 ). The NE-PER kit (ThermoFisher Scientific) was used for extraction of nuclear and cytoplasmic protein according to the manufacturer’s instructions.
Stat5a
Manufactured by Cell Signaling Technology
Sourced in United States
STAT5A is a transcription factor that plays a central role in cellular signaling pathways. It is involved in the regulation of gene expression in response to various extracellular signals.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
P stat5ay694, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
α tubulin antibody, by Merck Group (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Stat5b, by Cell Signaling Technology (1 mentions)
Flag hrp, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Actin hrp, by Merck Group (1 mentions)
Phospho stat5, by Cell Signaling Technology (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Las3000 luminescent image analyzer, by GE Healthcare (1 mentions)
Nfatc1, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
P erbb3, by Santa Cruz Biotechnology (1 mentions)
Total akt c67e7, by Cell Signaling Technology (1 mentions)
9 protocols using stat5a
1
Protein Expression Analysis by Western Blotting
2
Western Blot Analysis of Signaling Proteins
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Cells were washed with PBS and lysed in extraction buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF and protease inhibitor cocktails]. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore corporation, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBS-T [10 mM Tri-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and immunoblotted with antibodies against c-Fos (Santa Cruz Biotechnology, Dallas, TX, USA), NFATc1 (Santa Cruz Biotechnology), Phospho-STAT5 (Cell Signaling Technology), STAT5A (Cell Signaling Technology, Beverly, MA), IκB (Cell Signaling Technology), Flag-HRP (Sigma-Aldrich), Actin-HRP (Sigma-Aldrich), mouse-IgG-HRP (Abcam, Cambridge, UK) and rabbit-IgG-HRP (Abcam). Signals were detected with ECL solution (Millipore corporation) and analyzed using a LAS3000 luminescent image analyzer (GE Healthcare, Piscataway, NJ, USA).
3
Western Blot Analysis of Mammary Cells
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Mammary glands and cells were homogenized in ice-cold 50 mM Tris (pH 7.4), 100 mM NaF, 120 mM NaCl, 0.5% Nonidet P-40, 100 μM Na3VO4, and 1× protease inhibitor mixture (Roche), sonicated for 10 s on ice, and cleared by centrifugation at 4 °C, 13,000 × g for 20 min. Protein concentration was determined using the bicinchoninic acid assay (Pierce). Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 3% gelatin in TBS-T (Tris-buffered saline, 0.1% Tween-20) for 1 h, incubated in primary antibody in 3% gelatin overnight at 4 °C, washed with TBS-T, incubated in HRP-conjugated anti-rabbit (sc-2375, 1:10,000; Santa Cruz Biotechnology) or anti-mouse IgG (ab97240, 1:5000; Abcam), washed with TBS-T, and then developed using ECL substrate (Pierce). The following primary antibodies were used: P-ErbB3 (sc-135654, 1:500; Santa Cruz Biotechnology), P-MAPK/Akt/S6/Rab11 cocktail (1:1000; Cell Signaling Technologies), total Akt (C67E7, 1:1000; Cell Signaling Technologies), actin (1:2000; Cell Signaling), STAT5A and P-STAT5A/B (93585 at 1:500 and C11C5 at 1:500, respectively; Cell Signaling Technologies), and cleaved caspase 3 (D175, 1:500; Cell Signaling Technologies).
4
Immunoblotting Antibody Panel Analysis
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We used the following antibodies for immunoblotting: BCR-ABL1 (#2802), pBCR-ABL1 (#2861), BIM (#2819), cleaved CASPASE 3 (#9661), STAT5A (#9310), pSTAT5A (#9359), PARP (#9542), pERK (#4377), ERK (#9102), MCL-1 (#4572) (all from Cell Signaling Technology), Lyn (G-7, Santa Cruz, USA), pLYN (Epitomics, USA), BCL-2 (AbCam, UK) and β-actin (#AC-15, Sigma, USA). The antibody dilutions used were 1 in 1,000; except for β-actin (1 in 5,000). HRP-conjugated secondary antibodies were specific to rabbit (Sigma) or mouse IgG (Santa Cruz biotechnology). The protein bands on the membrane were visualized using the Western Lightning chemiluminescence reagent (pERKinElmer, USA).
5
Evaluation of HDAC Inhibitor Mechanism
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MPT0E028 and SAHA were synthesized by Professor Jing-Ping Liou. RPMI-1640 medium, FBS, penicillin, streptomycin, and all other tissue culture reagents were obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), LY294002 and all of the other chemical reagents were purchased from Sigma Chemical (St. Louis, MO, USA). The following antibodies were used: caspase 8, caspase 9, p-Akt(T308), Akt, p-mTOR, mTOR, p-GSK3β, p-eIF4E, p-p70S6K(T421/S424), p-p70S6K(T389), HDAC1, HDAC2, HDAC4, acetyl-α-tubulin, BID, STAT2, STAT4, STAT5A, STAT6 (Cell Signaling Technologies, Boston, MA, USA); PARP, HDAC6, HRP-conjugated anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Akt (S473) (Epitomics, Burlingame, CA, USA); caspase 6, caspase 7, p53, STAT1 (BD Biosciences PharMingen, San Jose, CA, USA); caspase 3 (Imgenex, San diego, CA, USA); acetyl-histone H3, STAT5B (Upstate Biotechnology, Lake Placid, NY, USA); Actin (Chemicon, Billerica, MA, USA). Trizol reagent was from Invitrogen (Carlsbad, CA, USA). Random primer and M-MLRT were purchased from Promega (Madison, WI, USA). Pro-Teq was from Protech (Taipei, Taiwan).
6
Western Blotting Quantification of Cell Signaling
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SDS-PAGE and western blots were performed following the standard protocols [31 (link)]. Antibody binding to bands was detected using an ECL detection system (Sage Creation). The primary antibodies used were specific to STAT5A (1:1000, #4807, Cell Signaling Technology, Danvers, MA, USA), DGCR8 (1:1000, #6914, Cell Signaling Technology), p-STAT5AY694 (1:1000, #4322, Cell Signaling Technology), p-STAT3Y705 (1:1000, #9145, Cell Signaling Technology), PCNA (1:1000, #13110, Cell Signaling Technology), GAPDH (1:10000, ab181602, Abcam, Cambridge, UK).
7
Phosphorylation Profiling of Kinase Targets
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To identify the relative levels of phosphorylation at the 46 kinase phosphorylation sites, the Proteome Profiler Human PhosphoKinase Array Kit (ARY003B, R&D Systems) was used in the DLD1 and HT29 cells transfected with the control and CIP2A-specific shRNAs according to the manufacturer’s instructions. The resulting spots were identified and images were quantified using the Image lab software (Bio-Rad) and Microsoft Excel software. The proteins were evaluated through western blotting, which were identified as follows: p53 (Abcam), p-p53 (S392) (Abcam), STAT5a (Cell Signaling, USA), p-STAT5a (Y694) (Cell Signaling, USA), Cyclin D1 (Cell Signaling, USA), Bax (Cell Signaling, USA), p21 (Cell Signaling, USA), p-AKT (T308) (Cell Signaling, USA), AKT (Cell Signaling, USA), p-ERK1/2 (Cell Signaling, USA), ERK1/2 (Cell Signaling, USA), and β-actin (Santa Cruz).
8
ChIP Analysis of STAT5a Binding in MC3T3-E1 Cells
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MC3T3-E1 cells were plated into 15 cm dishes (1.5 × 107 cells) at the densities described above in the Cell Culture section. ChIP was performed using antibodies against STAT5a (Cell Signaling Technology) (156 μg/mL) or rabbit IgG (Cell Signaling Technology) (2.5 mg/mL) using the ChIP-IT Express Enzymatic (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. DNA was amplified using primers flanking the putative STAT5a binding site (PROMO) within the GJA1 promoter.
9
Western Blot Analysis of Signaling Proteins
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Lysates were made using lysis buffer consisting of 50 mM Tris HCl at 7.5 pH, 250 mM NaCl, 0.5% (v/v) NP-40, and 5 mM EDTA, with proteases and phosphatases. A total of 50 µg of protein was loaded into SDS-PAGSE gels and transferred onto iBlot 2 nitrocellulose membranes (Invitrogen). The following antibodies were purchased from Cell Signaling Technology: RPSKB1, RPSKB2, RPSKA1, STAT5A, AKT1, p-AKT S473, p-S6, S6, p-eIF4E S209, and eIF4E. β-actin (A5316) was purchased from Sigma.
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