C2C12, 3T3-L1, and HEK293 cells were purchased from ATCC and maintained as instructed. Differentiation of C2C12 was performed as reported (Burattini et al., 2004 (link)). Transfection in C2C12 was performed using DharmaFECT1 as instructed (Thermo Scientific). 7,8-DHF was purchased from Tokyo Chemical Industry. HFD (40% kcal) was obtained from Research Diet. Human insulin was obtained from Eli Lilly. Other chemicals were purchased from Sigma-Aldrich. Antibodies against pAkt S473, IR, pIR Y1150/1151 (#3024), pAMPKa T172 (#2535), AMPKα (#2603), pACC S79 (#3661), ACC (#3676), pERK T202/ Y204 (#9106), ERK (#9102), pCREB S133 (#9198), CREB (#4820), and TrkB (4603) were purchased from Cell Signaling. Anti-Akt1 (sc5298) and anti-pTrkB Y706 (sc135645) antibodies were obtained from Santa Cruz Biotechnology. Anti-UCP1 antibody (ab155117) was obtained from Abcam. Anti-tubulin antibody (T6074) was obtained from Sigma-Aldrich.
Pcreb s133
Manufactured by Cell Signaling Technology
Sourced in United States
PCREB (S133) is a primary antibody that recognizes the phosphorylated form of the CREB (cAMP Response Element-Binding Protein) transcription factor at serine 133. This modification is associated with CREB activation and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti tubulin antibody t6074, by Merck Group (1 mentions)
Paccs79, by Cell Signaling Technology (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Dharmafect 1, by Thermo Fisher Scientific (1 mentions)
7 8 dhf, by Tokyo Chemical Industry (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
Human insulin, by Eli Lilly (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Ab156575, by Abcam (1 mentions)
Sc365422, by Santa Cruz Biotechnology (1 mentions)
Pyaps127, by Cell Signaling Technology (1 mentions)
Forskolin, by Merck Group (1 mentions)
Donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Fluoview fv 10i laser point scanning confocal microscope, by Olympus (1 mentions)
α tubulin, by Merck Group (1 mentions)
H3k9k14ac, by Cell Signaling Technology (1 mentions)
H3ps10, by Abcam (1 mentions)
Horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions)
Total h3, by Abcam (1 mentions)
Dc assay, by Bio-Rad (1 mentions)
10 protocols using pcreb s133
1
Molecular Mechanisms of Cell Differentiation
2
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed as previously described [36 (link)]. The following antibodies and reagents used: primary antibodies against Flag (#14793), PCNA (#13110), PKA C-α (#4782), CREB (#9197), pCREB (S133) (#9198), YAP (#14074) and pYAP (S127) (#13008) were purchased from Cell Signaling Technology, Inc. (diluted 1:1000; CST, MA, USA), a primary antibody against RAMP1 (#ab156575) was purchased from Abcam Plc (diluted 1:1000; Cambridge, UK), and a primary antibody against Gαi3 (#sc-365422) was purchased from Santa Cruz Biotechnology, Inc. (diluted 1:500; TX, USA); horseradish peroxidase (HRP)-linked anti-rabbit IgG (#7074) and HRP-linked anti-mouse IgG (#7076) secondary antibodies were purchased from CST (diluted 1:5000); and total protein-labelling No-Stain™ reagent (#A44449) was purchased from Thermo Fisher Scientific.
3
PGC-1β Activation and Signaling Pathway
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Forty-eight hours post-transduction with HA-Flag-PGC-1β adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 μM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1β (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).
4
Rapamycin-Induced CREB Phosphorylation
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ELT3 and LAM-derived cells were plated overnight on glass coverslips in 12-well tissue culture plates. Cells were serum-starved overnight, then treated with 20 nM rapamycin for 24 hours. Cells were rinsed with PBS twice, fixed with 4% paraformaldehyde for 30 minutes at 37°C, permeabilized with 0.2% Triton X-100, blocked in 3% BSA/PBS for 1 hour, and then incubated with primary antibody p-CREB (S133) (catalog 9198 from Cell Signaling Technology) in 1% BSA/PBS for 1 hour followed by secondary antibody donkey anti–rabbit IgG (H+L) (catalog A10042 from Invitrogen) for 1 hour at room temperature. Images were captured with a FluoView FV-10i Olympus laser point-scanning confocal microscope.
5
Immunoblotting Using Diverse Antibodies
6
Western Blotting for Protein Quantification
7
Western Blot Analysis of AKT Pathway
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Cell lysates were prepared in the cell lysis buffer [50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA and 1% NP-40 with one tablet (per 10 mL) of protease and phosphatase inhibitor (Life Technologies)]. After incubation on ice for 30 min, lysates were centrifuged at top speed for 10 min and supernatant were collected and proteins were quantified using Protein DC assay (BioRad). Proteins were running and separated on 4%–12% Bis-Tris Pre-cast Gel (Life Technologies) using MOPS buffer and transferred to PVDF membrane at 350 mA for 1.5 h. Membranes were blocked in 5% non-fat milk in TBST and incubated with primary antibody overnight. Primary antibody against pAKT (S473) [#3787, #4060], pAKT (T308) [#4056], pAKT2 (S474) [#8599], pan AKT [#2920], AKT1 [#2967], AKT2 [#3063], pCREB (S133) [#9198], pLATS1 (T1079) [#8654], pMOB1 (T35) [#8699], pYAP (S127) [#13008], pPKA substrate motif antibody [#9624], p110a [#4249], CREB [#4820], MOB1 [#13730], LATS1 [#9153], MST1 [#14946] and GAPDH [#5174] were from Cell Signaling Technology. MST2 [#ab52641] was from Abcam, and p110 (pT1061) polyclonal antibody was developed and purified by Cell Signaling Technology. HRP-conjugated secondary antibody was used at 1:5000 in 5% milk and membrane were developed using ECL solution and exposed to film. 7.5% phos-tag gel was used to resolve phosphor-p110a (T1061) (Wako Diagnostics/Chemicals # 192–18001).
8
Adiponectin Regulation of Lipolysis
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Immunoblots were conducted on 3 to 7 samples randomly chosen within each experimental cohort with antibodies against HSL (#4107 or #18381), p-HSL (S563) (#4139), p-HSL (S565) (#4137), p-HSL (S660) (#4126), Perilipin-1 (#9349), p-AMPKα (T172) (#2535), AMPKα (#2532), p-CREB (S133) (#9198), CREB (#9197), p-(Ser/Thr) PKA substrate (#9621), HA-tag (#3724), Adiponectin (#2789) and β-actin (#4970) from Cell Signaling (Denvers, MA); PHLPP1, (A300-660A), PHLPP2 (A300-661A) from Bethyl Laboratories, Inc (Montgomery, TX); PPARα (SC-9000) from Santa Cruz Biotechnology (Dallas, TX). Mouse serum was neutralized by an Adiponectin antibody (ab3455) from Abcam46 (link). All primary antibodies were used at a dilution of 1:1000. HRP-linked anti-rabbit IgG (NA934) and anti-mouse IgG (NXA931) were purchased from Amersham and used at a dilution of 1:5000. Alexa Fluor 594 anti-Rabbit IgG (A32754) was purchased from ThermoFisher and used at a dilution of 1:1000.
9
Western Blot Analysis of Signaling Pathways
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Cells were washed and lysed as previously described [23 (link)]. Total protein (10μg) was separated by SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare, Buckinghamshire, England). Antibodies used for immunoblotting included anti-pMEK1/2(Ser217/221), -MEK1/2 total, -ERK1/2(Thr202/Tyr204), -ERK1/2 total, PARP, -pAKT (S473), -AKT, pCREB (S133) and CREB (Cell Signalling Technology, Inc., MA, USA) and anti-GAPDH (Millipore, USA). Membranes were incubated with IRDye® 680LT or IRDye 800CW conjugated secondary antibodies and protein-antibody complexes visualized as previously described [48 (link)].
10
Cell Culture Protocols for Cancer and Normal Cells
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The HuCCT1 human bile duct carcinoma cell line was obtained from the Japanese Cancer Research Resources Bank and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). EHMES-1 human mesothelioma cells were cultured in RPMI-1640 medium supplemented with 10% FBS. Normal human renal proximal tubular epithelial cells were obtained from American Type Culture Collection (ATCC) and cultured in Renal Epithelial Cell Basal Medium (ATCC) supplemented with 0.5% FBS, 10 nM triiodothyronine, 10 ng/ml recombinant human epidermal growth factor, 100 ng/ml hydrocortisone hemisuccinate, 5 μg/ml recombinant human insulin, 1 μM epinephrine, 5 μg/ml transferrin, and 2.4 mM l -alanyl-l -glutamine. Recombinant human HGF (five amino acid deletion type) was prepared from the conditioned medium of CHO cells stably expressing human HGF. aMD5-PEG11 (Fig. 1A ) and aMD5 monomer peptide were synthesized as described previously13 (link). Anti-pERK (T202/Y204), -ERK, -pAKT (S473), -AKT, -pPRAS40 (T246), -PRAS40, -pSTAT3 (S727), -STAT3, -pCREB (S133), and -CREB antibodies were purchased from Cell Signaling.
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