Cells were lysed by NP-40 with protease inhibitors and phosphatase inhibitors; the proteins were separated via SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes, and then incubated with primary and secondary antibodies, respectively. Primary antibodies against JNK1/2/3 (YT2441) (1 : 1000) and phospho-JNK1/2/3 (phospho Thr183/Y185) (1 : 1000) were obtained from ImmunoWay Biotechnology (Newark, DE, USA). Primary antibodies against ERK (ab196883, 1 : 1000), p-ERK (ab50011, 1 : 5000), p38 (ab27986, 1 : 1000), and p-p38 (ab45381, 1 : 5000) were purchased from Abcam (Abcam, Cambridge, MA, United States). The JNK inhibitor SP600125 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The immunoreactive bands were detected with an enhanced chemiluminescence detection system from Pierce (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein band was analyzed with Quantity One software (Bio-Rad Laboratories, Life Science Research, CA, USA). Protein levels were normalized to that of the internal control β-actin purchased from Abcam (Cambrige, MA, USA) (ab8226, 1 : 1000).
Ab27986
Manufactured by Abcam
Sourced in United States
Ab27986 is a polyclonal antibody directed against the protein target. It is designed for use in various laboratory applications, such as Western blotting, immunohistochemistry, and immunofluorescence. The antibody is produced in rabbits and purified using protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab50011, by Abcam (1 mentions)
Ab45381, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab196883, by Abcam (1 mentions)
Sp600125, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab12172, by Abcam (1 mentions)
Ab58632, by Abcam (1 mentions)
Ab23981, by Abcam (1 mentions)
Aprotinin, by Beyotime (1 mentions)
Ab37168, by Abcam (1 mentions)
Mmp 13, by Bioss Antibodies (1 mentions)
Sc 2374, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab9483, by Abcam (1 mentions)
Ab64148, by Abcam (1 mentions)
Anti p akt, by Abcam (1 mentions)
4 protocols using ab27986
1
Western Blot Analysis of MAPK Pathway
2
Western Blot Analysis of Chondrocyte Markers
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The cells were extracted with lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM PMSF and 0.2% Aprotinin (Beyotime). After we measured the protein concentration, the equal protein samples were mixed with 5X sample buffer (Beyotime) and boiled. The proteins (30 µg) were resolved by 10% SDS-PAGE gel and transferred on PVDF membrane (Millipore, Hong Kong, China) by using the semi-dry transfer method. After blocking in 10% nonfat dried milk in TBST for 2 h, the blots were incubated with primary antibodies including HDAC4 (rabbit polyclonal 1:500, ab12172), p38 (rabbit polyclonal 1:500, ab27986), Runx2 (rabbit polyclonal 1:500, ab23981), Col10a1 (rabbit polyclonal 1:500, ab58632) (all from Abcam), p-p38 (rabbit polyclonal 1:500, bs-50486R), Mmp13 (rabbit polyclonal 1:500, bs-0575R) (both from Bioss) and GAPDH (rabbit polyclonal 1:1,000, ab37168; Abcam) at 4°C overnight. After washing by TBST, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (diluted 1:2,000, sc-2374; Santa Cruz Biotechnology) at room temperature for 1 h. Blots against GAPDH served as loading control.
3
Immunoblotting Analysis of Cellular Signaling
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Total extracted proteins were separated by electrophoresis and were subsequently transferred onto polyvinylidene fluoride membranes. After electrophoresis, the membranes were incubated with the below primary antibodies: anti-GAPDH (#ab9483; Abcam), anti-p38 (#ab27986; Abcam), anti-phosphorylated (p)-p38 (#ab236527; Abcam), anti-STAT3 (#ab68153; Abcam), anti-p-STAT3 (#b32143; Abcam), anti-AKT (#ab64148; Abcam), and anti-p-AKT (cat. no. ab8933; Abcam). Then, the membranes were incubated with relative secondary antibodies at room temperature for 2 h.
4
Western Blot Analysis of Retinal Proteins
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Total cellular and nuclear protein was extracted from retinas and quantified using bicinchoninic acid assay kit. Homogenate in 2 × sodium dodecyl sulfate (SDS) sample buffer was boiled for 5 min, and then equal amounts of protein (40 μg) from each sample were subjected to electrophoresis on a 10% (v/v) SDS-polyacrylamide gel. After proteins were electroblotted to a polyvinylidene difluoride membrane, the membrane was blocked with Phosphate-buffered saline containing 5% dried non-fat milk or 3% BSA at room temperature for 1 h, and incubated with indicated primary antibodies (anti-ATF4, 1:1000, SC-200, Santa Cruz Biotechnology, Dallas, TX; anti-ATF6, 1:1500, ab37149; anti-GRP78/BiP, 1:2000, ab21685; anti-pIRE1α, 1:1000, ab48187, Abcam, Cambrigde, MA; anti-Nrf2, 1:1000, SC-722; Santa Cruz Biotechnology, Dallas, TX; anti-p38, 1:500, ab27986; anti-p-p38, 1:1000, ab4822; anti-JNK, 1:1000, ab59227; anti-p-JNK, 1:2000, ab124956; anti-p65, 1:600, ab7970; anti-p-p65, 1:2000, ab86299; anti-SIRT1, 1:2000, ab12193, Abcam, Cambrigde, MA) at 4 °C overnight, followed by incubating with the goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody for 2 h. After incubation, membrane was washed three times, and the antigen-antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer, Akron, OH).
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