For SDS-PAGE analysis, total proteins in hucMSCs and exosomes were separated on 12% SDS-polyacrylamide gels and stained with Coomassie Blue. For Western blot assay, the proteins were electroblotted onto a nitrocellulose membrane after separating on 12% SDS-PAGE. The membrane was blocked and incubated with the primary antibodies followed by incubation with the horseradish peroxidase-coupled secondary antibody. The bands were visualized with ECL plus system from Amersham Pharmacia Biotech (Buckinghamshire, UK). The sources of primary antibodies were as follows: CD9 (Bioworld Technology, USA), CD81 (Epitomics, USA), β-actin (Bioworld Technology, USA), p-ERK1/2, T-ERK1/2, p-P38, P38 (Santa Cruz Biotechnology, USA), PCNA (Bioworld Technology, USA), and GAPDH (Shanghai KangChen Biotechnology, China).
T erk1 2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
T-ERK1/2 is a laboratory product that detects the total levels of extracellular signal-regulated kinase 1 and 2 (ERK1/2) proteins in cellular samples. It is a tool used for quantitative analysis of ERK1/2 expression.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
T akt, by Cell Signaling Technology (2 mentions)
Ecl plus system, by Cytiva (1 mentions)
Proliferating cell nuclear antigen (pcna), by Bioworld Technology (1 mentions)
β actin, by Bioworld Technology (1 mentions)
T akt, by Santa Cruz Biotechnology (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Antibody against foxo3a, by Cell Signaling Technology (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
T eif2α, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P perk, by Cell Signaling Technology (1 mentions)
P rps6, by Cell Signaling Technology (1 mentions)
Ki67 clone sp6, by Abcam (1 mentions)
K48 ub, by Cell Signaling Technology (1 mentions)
T mtorc1, by Cell Signaling Technology (1 mentions)
P yap, by Proteintech (1 mentions)
Grp78, by StressMarq Biosciences (1 mentions)
6 protocols using t erk1 2
1
Protein Profiling of hucMSCs and Exosomes
2
Molecular Pathways of Glucocorticoid Signaling
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Dexamethasone (DEX, CAS Number: 50-02-2), Corticosterone 21-acetate (CORT, 98%; CAS Number: 1173-26-8), were purchased from Sigma-Aldrich (USA). Human recombinant NGF was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Glucocorticoid receptor inhibitor RU-486 was purchased from Calbiochem (La Jolla, CA, USA). Specific antibodies towards p-FoxO3a, p-AKT, T-AKT, p-ERK1/2, T-ERK1/2, GAPDH and β-actin were obtained from Santa Cruz Biotechnology (Delaware Ave, Santa Cruz, USA). Specific antibody towards NPW was obtained from Biobyt (Wuhan, China). Antibody against FoxO3a was obtained from Cell Signaling Technology (Woburn, USA). Antibody against CRH was obtained from Signalway antibody (Pearland, Texas, USA). GFP-N1 and GFP-FoxO3a were kindly provided by Dr. Marten P. Smidt, University Medical Center, Utrecht, Holland. The primers of RPL-19, FoxO3a and NPW were obtained from Invitrogen Co. Ltd. (Guangzhou, China). FoxO3a-siRNA was purchased from GenPharma Co. Ltd. (Shanghai, China).
3
Antibody Panel for Cellular Signaling
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The following antibodies were used for IHC and Western immunoblotting: ATF4 (catalog 10835-1-AP) and SKP2 (catalog 15010-1-AP) from Proteintech; CHOP (catalog 2895), p-YAP (catalog 13008, S127), YAP (catalog 14074), p27 (catalog 3686), MYC (catalog 9402), p-RPS6 (catalog 4858, S235/236), t-RPS6 (catalog 2217), p-mTORC1 (catalog 5536, S2448), t-mTORC1 (catalog 2983), p-ERK1/2 (catalog 9101, T202/Y204), t-eIF2α (catalog 2103), p-AKT (catalog 9271, S473), t-AKT (catalog 4691), EIF4FBP1 (catalog 2855, T37/46), K48-Ub (catalog 8081), p-PERK (catalog 3179, T980), and PERK (catalog 3192) from Cell Signaling Technology; CYR61 (catalog E-AB-14920), CTGF (E-AB-12339), and aquaporin 2 (E-AB-30540) from Elabscience Biotechnology; CDK1 (catalog SC-54) and t-ERK1/2 (catalog SC-94) from Santa Cruz Biotechnology; α-tubulin (catalog T5168) and K63-Ub (catalog 05-1308) from MilliporeSigma; PCNA (catalog MS-106-P1ABX and, p-LATS1/2 (catalog PA5-64591, S809/S872) from Thermo Fisher Scientific; Oct4 (catalog NB100-2379) from Novus Biologicals; Sox2 (catalog ab97959), vimentin (catalog ab92547), Ki-67 (clone SP6), p-eIF2α (catalog ab32157, S51), BIM (catalog ab32158), and TAZ (catalog ab224239) from Abcam; GRP78 (catalog A0241) from StressMarq Biosciences; and nestin 1 (monoclonal rat-401s) from DSHBU Iowa.
4
Western Blot Analysis of Signaling Proteins
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Total cell lysates were prepared with protein lysis buffer (Intron Biotechnology, Seoul, Korea) supplemented with 1× protease inhibitor cocktails and PMSF (Sigma-Aldrich, St. Louis, MO, USA). The lysates were suspended with sample buffer containing sodium dodecyl sulfate (SDS) and boiled for 7 min for denaturation. Proteins were separated on 8% or 12% SDS-polyacrylamide gels electrophoresis, and then transferred onto a polyvinylidene fluoride (PVDF) membranes. The membrane was incubated with primary antibodies in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for overnight at 4 °C. Primary antibodies for p-ERK1/2, t-ERK1/2, p38, p-JNK, t-JNK, AKT, and β-actin were purchased from Santa Cruz biotechnology Inc. (Santa Cruz, CA, USA). p-p38 and p-AKT antibodies were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). The membrane washed with TBS-T to remove primary antibody, followed by incubation with specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 2 h. The signals were visualized using enhanced chemiluminescence (ECL; Abclon, Seoul, Korea) and an Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan).
5
Prefrontal Cortex Immunofluorescent Analysis
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The prefrontal cortices of mice were analyzed via immunofluorescent assays. Coronal sections (5-μm thickness) were prepared and immersed in 4% PFA for 30 min, followed by three washes in TBST. After blocking non-specific binding via 1.5% BSA for 1 h, sections were then incubated with corresponding primary antibodies overnight at 4 °C, as follows: GFAP (1:50; Proteintech, Wuhan, Hubei, China) and P-ERK1/2 (1:100; Beyotime, Shanghai, China); GFAP and GLUT3 (1:100; Santa Cruz, Dallas, Texas, USA); NeuN (1:50; Proteintech, Wuhan, Hubei, China); P-ERK1/2; and GFAP and T-ERK1/2 (1:100; Santa Cruz, Dallas, Texas, USA). After washing three times with TBST, the secondary antibody mix with FITC-conjugated goat anti-mouse IgG (1:200; Bioss, Beijing, China) and Cy3-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China) was added to cover the tissue and was incubated at room temperature for 1 h in the dark. Sections were then incubated with DAPI at room temperature for 5 min. Fluorescent images were obtained, and image analysis was applied to quantify immunoreactive signals.
6
Protein Expression Analysis of Combinatorial Treatments
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200.000 cells were cultivated in a 12-well format and treated with IC50 concentrations for single or combinational treatment for 48 hours. Proteins were isolated using RIPA lysis buffer supplemented with cOmplete Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche Diagnostics). Protein concentration was measured using Bradford reagent (Sigma-Aldrich, St. Louis, Missouri, USA). 30 µg of protein was loaded and separated on a 10% polyacrylamid gel in a Tris-Glycin-SDS buffer. Proteins were then blotted onto nitrocellulose membranes using Turboblot (Biorad, Hercules, California, USA). Membranes were blocked in 5% bovine serum albumin (Sigma-Aldrich) diluted in TBS-T and incubated with primary antibodies overnight at 4° C. Primary antibodies used were P-AKT Ser473 (Cell Signaling, #4060), T-AKT (Cell Signaling, #), P-ERK1/2 Tyr204 (Santa Cruz, sc-101761), P-ERK1/2 Thr202/Tyr204 (Cell Signaling, #9101), T-ERK 1/2 (Santa Cruz, sc-93/sc-154). Visualization was performed by using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Laboratories, Dallas, Texas, USA) and the enhanced chemiluminescence detection system (Biorad) on a ChemiDoc XRS+ Imaging device (Biorad).
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