PEDV-, TGEV- and PEDV + TGEV-infected and mock-infected IPEC-J2 cells at 1 and 24 hpi were harvested by 0.25% trypsinisation, then fixed and permeabilised in FACS permeabilisation buffer II (BD Pharmingen, USA), washed 3 times with 0.01 M PBS and incubated with a 1% solution of BSA (30 min, RT). Then, cells stained with the primary antibodies anti-ERK1/2 antibody, anti-ERK1/2 (phospho T202 + Y204) antibody, anti-p38 antibody, anti-p38 (phospho T180 + Y182) antibody, anti-JNK1 + JNK2 antibody and anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (Abcam, Hong Kong) were incubated overnight at 4 °C. Subsequently, cells were washed 3 times with 0.01 M PBS and the PE-conjugated goat anti-rabbit IgG2b (Invitrogen, USA) was added (1 h, RT). The negative control cells were treated in an identical manner, except the primary antibodies were omitted. Cells were then washed, and 1 × 104 cells were analysed by flow cytometry for the mean fluorescence intensity (MFI). The results are expressed as the mean fluorescence intensity ratio. The mean fluorescence intensity ratio was calculated using the formula: (MFIinfection phospho protein/MFIinfection total protein)/(MFIcontrol phospho protein/MFIcontrol total protein).
Anti erk1 2 antibody
Manufactured by Abcam
Sourced in Hong Kong, United States
Anti-ERK1/2 antibody is a laboratory research reagent used to detect and quantify the expression levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in biological samples. It is a specific antibody that binds to these proteins, allowing for their identification and measurement using various analytical techniques.
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Lab products found in correlation
Anti perk1 2 antibody, by Abcam (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti p38, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti ras antibody, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Anti p erk1 2, by Abcam (1 mentions)
Anti sp1, by Abcam (1 mentions)
Ecl kit, by Affinity Biosciences (1 mentions)
Anti p p38, by Proteintech (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Anti egfr antibody, by Abcam (1 mentions)
Anti akt antibody, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Anti stat3 antibody, by Abcam (1 mentions)
6 protocols using anti erk1 2 antibody
1
Activation of MAPK Signaling Pathways in IPEC-J2 Cells Infected with PEDV and TGEV
2
Western Blot Analysis of Signaling Proteins
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Total protein was extracted using RIPA lysis buffer (Sigma) containing protease and phosphatase inhibitors (Beyotime, China) and quantified using the BCA kit (Beyotime, China). Proteins (30 mg of total protein) were separated by 12% or 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in TBST containing 5% nonfat milk, and incubated overnight at 4°C with various primary antibodies, including anti-FGF2 antibody (Absin); anti-GRB2 antibody (Abcam); anti-ERK1/2 antibody (CST); anti-pERK1/2 antibody (CST); anti-Ras antibody(Abcam); anti-β-Actin antibody(CST); anti-GAPDH antibody(Abcam). Next, it was incubated with an HRP-conjugated goat anti-rabbit IgG (CST). The values of band intensities were detected by ECL kit (Beyotime, China).
3
Western Blot Analysis of Immune Signaling
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THP‐1 or Raw264.7 cells were lysed with RIPA buffer mixed with protease and phosphatase inhibitors (100:1). Protein concentrations were quantified. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) were used to separate equal amounts of protein, which were then transferred to PVDF membranes (Millipore, Burlington, MA). After being blocked in 5% skimmed milk powder for 1 h at room temperature, the membranes were treated with the following primary antibodies overnight at 4°C: anti‐SP1 (Abcam), anti‐PSRC1 (GeneTex), anti‐ANXA2 (CST), anti‐p‐STAT3 (CST), anti‐STAT3 (CST), anti‐ERK1/2 antibody (Abcam), anti‐p‐ERK1/2 (Abcam), anti‐P38 (CST), anti‐p‐P38 (Proteintech) and anti‐β‐tubulin (Fude Biotech). The membranes were incubated wit secondary antibodies (Fude Biotech). Using an ECL kit, the protein bands were seen (Affinity, China) and analysed by ImageJ (National Institutes of Health, USA).
4
Evaluating EGFR, STAT3, AKT, and ERK Signaling
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Anti-EGFR antibody, anti-STAT3 antibody, anti-AKT antibody, and anti-ERK1/2 antibody were purchased from Abcam. Fetal calf serum (FCS) was obtained from Gibco (USA). Phospho-STAT3, phospho-AKT, and phospho-ERK1/2 antibodies were purchased from CST company (USA). Bovine Serum Albumin (BSA) and PVDF membranes were purchased from Beyotime Biotechnology (Shanghai, China). Cell culture plates were purchased from Corning (New York, USA). DMEM, hypoxanthine-aminopterin-thymidine (HAT), and HT were purchased from Invitrogen (California, USA). The low-fluorescence PVDF membrane was purchased from Bio-Rad Laboratories. Unless otherwise specified, reagents were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA). Animal Experiments were performed under a project license (No.: 20200506) granted by animal ethics committee of First Hospital of Shanxi Medical University, in compliance with national guidelines for the care and use of animals.
5
Comprehensive Western Blot Analysis
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Western blot analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-FOXM1 antibody (Santa Cruz), anti-pAKT antibody (Peprotech, USA), anti-AKT antibody (Cell Signaling Technology, Beverly, MA, USA), anti-pGSK3β antibody (Proteintech) at 1:1000, anti-Snai1 antibody (Cell Signaling Technology), anti-pIGF1R antibody (Abcam, Cambridge, UK), anti-IGF1R antibody (Abcam, Cambridge, UK), anti-E-cadherin antibody (BD Biosciences, USA), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology), anti-pERK1/2 antibody (Abcam), anti-ERK1/2 antibody (Abcam), anti-HIF1α antibody (Novus Biologicals, USA), anti-ETS1 antibody (Abcam) and anti-β-actin antibody (Sigma, St. Louis, MO,USA).
6
Protein Profiling of N-ADSCs and ADSCs
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Total protein was isolated from N-ADSCs and ADSCs cultured under high glucose for 48 h in vitro, and the muscle tissues of db/db mice denervated limbs injected with ADSCs and N-ADSCs, respectively. The total protein in tissue samples or cell lysates was quantified, electrophoresed, and transferred to PVDF membranes that were probed with appropriate antibodies at 4 °C overnight as follows: anti-Akt and P-AKT antibody, anti-PI3K and P-PI3K antibody, anti-P38 and P-P38 antibody, anti-eNOS and P-eNOS antibody, anti-NF-κB and P-NF-κB antibody, anti-JNK antibody, anti-ERK1/2 antibody, and anti-β-actin antibody (1:500; Abcam). The Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA) was used to quantify the relative integral density of the immunoreactive bands.
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