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5 protocols using anti p p38

1

Western Blotting Analysis of Apoptosis and Inflammation Signaling

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Western blotting analysis was carried out as previously described [25 (link)]. The following antibodies were used: anti-Bcl-2 (#ab182858), anti-Bax (#ab182733), anti-Bcl-xl (#ab32370), anti-p-P65 (#ab76302), anti-P65 (#ab32536), anti-p-IκBα (#ab133462), anti-IκBα (#ab32518) (Abcam Cambridge, MA, USA), anti-FXR (#sc-25309), anti-TLR4 (#sc-293072) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-p-P38 (#4511), anti-P38 (#8690), anti-p-JNK1/2 (#4668), anti-JNK1/2 (#9252), anti-CCL2 (#66272-1-Ig), and anti-β-actin (#66009-1-Ig) (Proteintech, Wuhan, China).
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2

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).
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3

Comprehensive Western Blot Methodology

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Western blot was performed as previously described20 (link). Antibodies used in this study were PDLIM1 (Affinity, DF3003), α-SMA (Affinity, AF1032), COL1A1 (Cell Signaling Technology, #91144), CTCF (Millipore, 07-729), TNF-α (Affinity, AF7014), IL-6 (Affinity, DF6087), p65(Affinity, AF5006), anti-p-Smad2/3 (Abcam, ab254407), anti-Smad2/3 (Abcam, ab202445), anti-p-ERK (Abcam, ab201015), anti-ERK (Abcam, ab184699), anti-p-P38 (Proteintech, 28796-1-AP), anti-P38 (Proteintech, 66234-1-Ig), anti-p-JNK (Proteintech, 80024-1-RR), anti-JNK (Proteintech, 66210-1-Ig), GAPDH (Cell Signaling Technology, #2118). Western blot analysis was performed on at least three independent biological replicates.
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4

CHMP4A Knockdown and Overexpression Analysis

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ShCHMP4A was cloned into pSIH‐H1 puro (System Biosciences). The target sequence of CHMP4A short hairpin RNA (shRNA) was 5′‐GCCTAGTGTACCTTCTACT‐3′. FLAG‐tagged CHMP4A eukaryotic expression vector was constructed by inserting PCR‐amplified CHMP4A fragment into pcDNA3.0‐FLAG vector (Invitrogen).
Trizol reagent was purchased from Invitrogen. AMG487 (CXCR3 antagonist) and maraviroc (CCR5 antagonist) were purchased from Tocris. RNase A and GSK‐LSD1 (LSD1 inhibitor) were obtained from Sigma Aldrich. Anti‐dsRNA (J2) was purchased from English and Scientific Consulting Kft. Anti‐CHMP4A was purchased from Sino Biological. Anti‐Flag, Anti‐LSD1, and Anti‐β‐actin were purchased from Santa Cruz Biotechnology. Anti‐p70S6k, Anti‐p‐p70S6K, Anti‐mTOR, Anti‐p‐mTOR, Anti‐Akt, Anti‐p‐Akt, Anti‐ERK, and Anti‐p‐ERK were purchased from Cell Signaling Technology. Neutralizing Anti‐IFNβ, Anti‐PTEN, Anti‐P53, Anti‐P38, and Anti‐p‐P38 were purchased from Proteintech. Human IFNβ ELISA Kit was obtained from CUSABIO.
Whole blood samples, anticoagulated by sodium citrate, were collected from six healthy volunteers (50% male, medium age of 32 years, ranging from 25 to 42 years). The study was approved by the Ethics Committee of the Fourth Medical Center of the Chinese PLA General Hospital. Written informed consent was obtained from all volunteers.
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5

Hepatoprotective Mechanisms of Sal and Ani

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Sal and anisomycin (Ani) were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and was diluted in normal saline and stored away from light at −20°C. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) microplate test kits were acquired from the Jiancheng Bioengineering Institute (Jiancheng Biotech, Nanjing, China). IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Anogen (Ontario, Canada). Oligonucleotide primers were synthesized by Generay (Shanghai, China). The PrimeScript RT Reagent Kit and SYBR Premix Ex Taq were purchased from TaKaRa Biotechnology (Dalian, China). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit was obtained from Roche (Roche Ltd., Basel, Switzerland).
The primary antibodies used in this study were anti-Bax, anti-caspase 3, anti-caspase 9, anti-Beclin-1, anti-P62, anti-JNK, anti-P38, anti-p-P38 (Proteintech, Chicago, IL, USA), anti-TNF-α, anti-Bcl-2, antimicrotubule-associated protein 1 light chain 3 (LC3), antimammalian target of rapamycin (mTOR), anti-p-mTOR, anti-Akt, anti-p-Akt, anti-S6, anti-p-S6 (Cell Signaling Technology, Danvers, MA, USA), anti-IL-6, anti-p-JNK (Antibody Revolution, San Diego, CA, USA), anti-ERK, and anti-p-ERK (Signalway Antibody, College Park, MD, USA).
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