Western blots were performed as previously described elsewhere. Membranes were incubated with the following primary antibodies: anti‐MYBBP1A (Proteintech #14524‐AP, Rosemont, IL, USA ), anti‐PGC1α (Abcam #ab54481, Cambridge, UK), anti‐SGLT1 (Abcam #ab14685), anti‐p38 MAPK (Cell Signaling #9212, Danvers, MA, USA), and anti‐phospho‐p38 MAPK (T180/Y182) (Cell Signaling #9215). α‐Tubulin (Sigma #T9026) was used as a loading control. Horseradish peroxidase‐labeled rabbit anti‐mouse (Abcam #ab 97046) and goat anti‐rabbit (Abcam #ab 97051) secondary antibodies were used. The proteins were detected using an ECL detection system (Amersham Biosciences) and Bio‐Rad ChemiDoc XRST (Berkeley, CA, USA).
Anti p38 mapk
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-p38 MAPK is a laboratory reagent produced by Cell Signaling Technology. It is a primary antibody that specifically binds to and detects the p38 mitogen-activated protein kinase (MAPK) protein. p38 MAPK is a key signaling molecule involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho p38 mapk, by Cell Signaling Technology (60 mentions)
Anti erk1 2, by Cell Signaling Technology (36 mentions)
Anti jnk, by Cell Signaling Technology (31 mentions)
Anti p p38 mapk, by Cell Signaling Technology (29 mentions)
Anti akt, by Cell Signaling Technology (26 mentions)
Anti phospho jnk, by Cell Signaling Technology (20 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (16 mentions)
Anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (15 mentions)
Anti phospho akt, by Cell Signaling Technology (15 mentions)
Anti sapk jnk, by Cell Signaling Technology (14 mentions)
Anti erk, by Cell Signaling Technology (12 mentions)
Anti nf κb p65, by Cell Signaling Technology (12 mentions)
Anti gapdh, by Cell Signaling Technology (11 mentions)
Anti β actin, by Merck Group (11 mentions)
Anti p erk1 2, by Cell Signaling Technology (10 mentions)
Anti β actin, by Cell Signaling Technology (10 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (9 mentions)
Anti bax, by Cell Signaling Technology (9 mentions)
Anti bcl 2, by Cell Signaling Technology (9 mentions)
Anti p44 42 mapk erk1 2, by Cell Signaling Technology (9 mentions)
153 protocols using anti p38 mapk
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of Signaling Proteins
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BM cells were lysed in a RIPA Buffer (Biosesang, Seongnam, Republic of Korea) including a protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), and they were sonicated on ice. After centrifugation, clear cell lysates were acquired and assayed for protein concentration. Following denaturation, 40 µg of protein was separated on a 4–12% SDS-PAGE gel (Invitrogen, Waltham, MA, USA) at 150 V for 90 min and transferred to a methanol-soaked PVDF membrane (Invitrogen) at 40 V for 100 min. The membranes were blocked with 3% bovine serum albumin for 1 h and then incubated at 4 °C overnight with anti-phopho-SAPK/JNK (Thr183/Tyr185) (#9255, Cell Signaling Technology, Danvers, MA, USA), anti-phopho-p38 MAPK (Thr180/Tyr185) (#9216, Cell Signaling Technology), anti-SAPK/JNK (#9252, Cell Signaling Technology) and anti-p38 MAPK (#9212, Cell Signaling Technology) Abs. The membranes were then incubated with anti-mouse or anti-rabbit IgG Abs conjugated to horseradish peroxidase (HRP) at room temperature for 1 h. After stripping at 55 °C for 30 min, they were incubated at room temperature for 1 h with anti-β-actin Abs (Santa Cruz Biotechnology, Dallas, TX, USA) and subsequently with anti-mouse HRP Abs (Cell Signaling Technology). The protein expression levels were normalized to the corresponding β-actin levels.
3
Signaling Pathway Analysis in Neuronal Cells
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B-27, L-glutamic acid, deoxyribonuclease (DNase), fluorescent dye, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), soybean trypsin inhibitor, fluo-8, and poly-l-lysine (PLL) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-p-p38 MAPK (p-p38), anti-p-JNK, anti-p-p44/42 MAPK (p-ERK1/2), anti-p38 MAPK, anti-JNK, anti-ERK1/2, and anti-rabbit/mouse IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Donkey anti-rabbit/mouse secondary antibodies and Alexa Fluor 488 were obtained from Jackson ImmunoResearch Laboratories, Inc., (PA, USA). Fetal bovine serum, neurobasal medium, RPMI 1640 medium, and trypsin were purchased from Gibco (Gaithersburg, MD, USA). The Gαq inhibitor YM 254,890 was obtained from Wako Pure Chemical Industries (Osaka, Japan). L-741,626, SCH 23390, NMDA, and PP2, LY 23,959 were purchased from Tocris Bioscience (Ellisville, MO, UK). YM 254,896 and PP2 were dissolved in 2% DMSO, L-741,626 was dissolved in 25% DMSO, SCH 23390, NMDA and LY2359549 were dissolved in sterilized saline.
4
Flow Cytometry Immunophenotyping Protocol
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Flow cytometry: commercial antibodies anti-CD86-FITC (clone 2231 FUN-1), CD274 (PD-L1)-FITC (clone MIH1), CD273 (PD-L2)-PE (clone MIH-18), HLA-DR-PE-Cy7 (clone L243), IFN-γ-FITC (clone 4SB3) were purchased from BD Biosciences; CD83-PerCP-Cy5.5 (clone HB15a) was purchased from Beckman Coulter; CD80-FITC (clone MAB104), CD40-PerCP-eFluor710 (clone 5C3), CD1a-PE-Cy7 (clone HI149) and CD4-PE-Cy7 (clone RPA-T4) were purchased from eBioscience; TLR2-FITC (clone T2.5), TIM-3-PE (clone F38-2E2), IL-10-PE (clone JES3-9D7), KI-67-PE (clone Ki-67) were purchased from BioLegend; CD14-PE-DL594 (clone MEM-15), CD11c-APC (clone BU15), CD3-AF700 (clone MEM-57), CD8-PE-Dy590 (clone MEM-31) were purchased from Exbio; CD85k (ILT-3)-PE (clone 293623), CD85d (IL-T4)-FITC (clone 287219) were purchased from R&D Systems. For western blot, anti-p-p38 MAPK, anti-p-ERK1/2, anti-p-JNK/SAPK, anti-p-IκB-α, anti-IDO, anti-p-mTOR, anti-p-STAT3, anti-p-p70S6K, anti-p38 MAPK, anti-ERK1/2, anti-JNK/SAPK and anti-STAT3 Ab were purchased from Cell Signaling Technology; anti-actin was from BioLegend.
5
Immunoprecipitation and Western Blot for α7 nAChR
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Immunoprecipitation (IP) or co‐IP of the α7 nAChR was performed as described 19 . Briefly, a co‐IP of the α7 nAChR protein complex was obtained from 500 μg cell membrane protein fraction using 5 μg of the C‐20 antibody (Santa Cruz, Dallas, TX, USA) 19 . Protein complexes associated with the co‐IP were captured using a Protein G Dynabeads (Thermo Fisher). For western blot detection, 100 μg of protein was loaded into each lane of an SDS/PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Thermo Fisher) for immunoblot detection using the following antibodies: anti‐Gαs (Rabbit) (New East Bioscience), anti‐Gαq (Rabbit) (New East Bioscience), anti‐Gαi (Rabbit) (New East Bioscience), anti‐Gβ (T‐20) (Santa Cruz), anti‐phospho‐p38MAPK (Thr180/Tyr182), anti‐p38MAPK, anti‐phospho‐CDC42/Rac1 (Ser 71), and anti‐GAPDH (Cell Signaling, Danvers, MA, USA). Horseradish peroxidase (HRP)‐conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Immunoblots were visualized using SuperSignal West Pico Chemiluminescence (Thermo Fisher) via a G:Box imaging system and GeneSYS software (Syngene, Fredrick MD, USA). SeeBlue protein standard (Thermo Fisher) was used as a molecular weight marker.
6
Western Blot Analysis of Cellular Signaling
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Protein samples from cultured cells were prepared by direct lysis in 5× SDS sample buffer, followed by heating at 95 °C for 5 min. Samples were resolved on 8–12% SDS–polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5% (w/v) dried milk in Tris-buffered saline/Tween (TBST) for 1 h at room temperature, after which they were incubated with primary antibody, followed by the correct horseradish-peroxidase-conjugated secondary antibody. Blots were developed using Western Bright ECL substrate (Advansta) or Immobilon Western chemiluminescent substrate (Millipore) using a Bio-Rad Gel Doc. Antibodies used were anti-HIF-1α (catalogue no. 14179), anti-IκB-α (9242), anti-phospho-NF-κB-p65 (3033), anti-NF-κB-p65 (8242), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (catalogue no. 9212), anti-phospho-ERK (9101), anti-ERK (4695), anti-phospho-p70-S6K (catalogue no. 9205), anti-p70-S6k (9202), all purchased from Cell Signaling Technology, anti-IL-1β (R&D Systems, AF401-NA), anti-β-actin (Sigma-Aldrich, AC-74), anti-FLAG (Sigma-Aldrich, F1804), L-2HGDH (Antibody Genie, CAB7996), and D-2HGDH (Antibody Genie, CAB16213). anti-HIF-1α from Novus (catalogue no. NB100-449) was used for human blots. Secondary antibodies used were horseradish-peroxidase-conjugated anti-mouse IgG, anti-goat IgG and anti-rabbit IgG (Jackson ImmunoResearch).
7
Inhibition of RAGE-Mediated Inflammation
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Dulbecco’s modified Eagle medium and penicillin–streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum was provided by Hyclone (Logan, UT, USA). Monoclonal rabbit antibodies, including anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p65, anti-phospho-p65, NLRP3, and cleaved caspase-1 were obtained from Cell Signaling Technology (Boston, MA, USA). Receptor for AGE (RAGE) monoclonal rabbit antibody was purchased from AbCam (Cambridge, MA, USA). HRP-marked anti-β-actin antibody was supplied by Biorad (San Diego, CA, USA). 4′-Methoxyresveratrol (3,5-dyhydroxy-4′-methoxylstilbene) was purchased from Great Forest Biomedical (Hangzhou, China). BSA was obtained from ABCONE (Shanghai, China). KI and acetic acid were obtained from Sangon Biotech (Shanghai, China). Methylglyoxal, 2’,7’-dichlorodihydrofluorescein diacetate and other reagents were purchased from Sigma (St. Louis, MO, USA).
8
Western Blot Analysis of Signaling Proteins
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Cells were lysed in RIPA buffer. Total proteins (50–150 μg) were resolved under reducing conditions by 7–10% SDS-PAGE and transferred to Immobilon-FL membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked in TBS containing 0.1% Tween 20 (TBS-T) and 5% milk for 1 hr at 25°C and then incubated overnight at 4°C with the following primary antibodies: anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-ERα (Ser118) (Merck Millipore), anti-ERK2, anti-Bek (C-17), anti-ERα, anti-lamin B, anti-E-cadherin (Santa Cruz Biotechnology) and anti-tubulin (Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich) for 1 hr at 25°C. Bound antibody was detected by enhanced chemiluminescence detection reagents (Pierce Biotechnology Inc., Rockford, IL, USA), according to manufacturer's instructions. Tubulin served to estimate the protein equal loading. Densitometric analysis was performed with Quantity One Program (Bio-Rad Laboratories S.r.l., Segrate, MI, Italy).
9
Antibody Detection of Cell Signaling
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Antibodies used were anti-RIPK1 (BD Biosciences, San Jose, CA, USA, #610459), anti-actin (MP Biomedicals, Solon, OH, USA, #69100), anti-IκB-α (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-371), anti-hRIPK3 (ThermoFisher Scientific Pierce, Waltham, MA, USA, PA1-41533), anti-mRIPK3 (Sigma-Aldrich, St Louis, MO, USA, #R4277 and IMGENEX, San Diego, CA, USA,, IMG-5523-2), anti-hMLKL (Genetex, Irvine, CA, USA, GTX107538), anti-mMLKL (Millipore, Billerica, MA, USA, #MABC604 and Abgent, San Diego, CA, USA, AP14272b-ev), anti-phospho-hMLKL (Abcam, Milton, Cambridge, UK, #187091), anti-hFADD (BD Biosciences, 610399), anti-mFADD (Millipore, 05-486), anti-thiophosphate ester (Epitomics, Burlingame, CA, USA, #2686-1), anti-Braf (ThermoFisher scientific, MA5-15495), anti-HPK1 (Cell Signaling, Danvers, MA, USA, #4472), anti-Hsp90 (Santa Cruz Biotechnology, sc-7947), anti-p38MAPK (Cell Signaling, #9212), anti-ERK1/2 (Cell Signaling, #9102), anti-Flag-HRP (Sigma-Aldrich, St Louis, MO, USA, #A8592) and anti-tubulin-HRP (Abcam, #ab21058). Secondary antibodies used were HRP-conjugated secondary antibodies against mouse, rabbit or rat immunoglobulin (GE Healthcare, Little Chalfont, Amersham, UK).
10
Cardamonin Inhibits NF-κB Signaling
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Cardamonin was purchased from Sigma-Aldrich (St Louis, MO, USA). BAY 11-7082 and MG132 and NAC were purchased from Beyotime Biotechnology (Haimen, China). TNF-α, SP600125, SB203580 were from PeproTech Inc. (Rocky Hill, NJ, USA). Cell Counting Kit-8 was from Dojindo Molecular Technologies (Kumamoto, Japan). Anti-NF-κB p65, anti-phospho-p65 anti-CDK4, anti-p21, anti-PARP, anti-JNK, anti-p38 MAPK, anti-phospho-JNK and anti-phospho-p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).
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