At 0, 24 and 48 hours, NPCs were collected and lysed in lysis buffer (Beyotime) on ice using a western and IP cell lysis kit. Then, the protein extracts were collected by centrifugation at 15 000 g for 15 minutes at 4°C. Protein concentrations of cell lysates were tested using an enhanced BCA protein assay kit (Beyotime). After protein transfer, the membranes were blocked with nonfat milk and then incubated overnight at 4°C with rat antibodies against LC3B (L7543, 1:1000, Sigma), p62 (sc‐48402, 1:500, Santa Cruz, USA), p16 (ab51243, 1:1000, Abcam), p21 (ab218311, 1:1000, Abcam), p53 (sc‐126, 1:500, Santa Cruz), PARKIN (ab77924, 1:200, Abcam), PINK1 (ab23707, 1:500, Abcam), COX IV (ab202554, 1:1000, Abcam) and β‐actin (8H10D10, 1:1000, CST). After three washes, the membranes were incubated using peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Finally, the proteins were visualized using the enhanced chemiluminescence method following the manufacturer's instructions (Amersham Biosciences).
Ab218311
Manufactured by Abcam
Sourced in United Kingdom
Ab218311 is a lab equipment product. It serves a core function within the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab32042, by Abcam (2 mentions)
Ab134175, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab185002, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Sc 48402, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence method, by Cytiva (1 mentions)
Ab23707, by Abcam (1 mentions)
Ab51243, by Abcam (1 mentions)
Ab77924, by Abcam (1 mentions)
Ab202554, by Abcam (1 mentions)
Sc 126, by Santa Cruz Biotechnology (1 mentions)
L7543, by Merck Group (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Ab140130, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by ZSGB-BIO (1 mentions)
6 protocols using ab218311
1
Autophagy and Senescence Markers Analysis
2
Evaluating Protein Expression Changes
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Following treatment with the selected compounds, the cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA kit. Standard western blot was used to evaluate total and phosphorylated protein expression levels. The antibodies used in this study were as follows: anti-Akt (1:1,000; AbSci, cat. no. AB 4685), anti-phospho-Akt (1:1,000; Ser473, AbSci, cat. no. AB 4060; Thr308, AbSci, cat. no. AB11055), anti-pro caspase-3 (1:1,000; AbSci, cat. no. AB9665), and p21 (1:1,000; Abcam, ab218311), Cyclin D1 (1:1,000; Abcam, ab134175), Bcl-2 (1:1,000; Abcam, ab185002), Bax (1:1,000; Abcam, ab32503), cleaved caspase 3 (1:1,000; Abcam, ab32042), and GAPDH (1:1,000; Abcam, ab181602).
3
Western Blot Analysis of Apoptosis Regulators
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Proteins were extracted with a Nuclear and Cytoplasmic Protein Extraction kit (catalogue no. P0028, Beyotime, Shanghai, China). Cell protein lysates were separated by 10% sodium salt (SDS)-polyacrylamide gel electrophoresis (PAGE) and then electroblotted from the gels onto polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics, Mannheim, Germany). After blocking the membrane with 5% nonfat milk powder and 0.1% Tween 20 in PBS for 1 h, the membrane was incubated with primary antibodies specific to GPX2 (catalogue no. ab140130), p21 (ab218311), Cyclin D1 (ab134175), Bcl-2 (ab185002), Bax (ab32503), cleaved caspase 3 (ab32042), and GAPDH (ab181602); all of which were acquired from Abcam (Cambridge, UK; the dilution for all was 1 : 1000). After extensive washing with blocking solution, blots were exposed to horseradish peroxidase-conjugated goat anti-rabbit IgG (catalogue no. ZB-2301, Zsbio, Beijing, China; 1 : 2500 dilution). Finally, the protein bands were imaged using an enhanced chemiluminescent (ECL) substrate (Merck Millipore, Hong Kong, China).
4
Western Blot Analysis of Hepatocellular Carcinoma
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Total proteins from HCC cells were extracted using radioimmunoprecipitation assay
lysis buffer and qualified by a bicinchoninic acid assay detecting kit (Thermo
Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were boiled at 100°C in
sodium dodecyl sulfate (SDS) sample buffer for 5 min and separated with
SDS-polyacrylamide gel electrophoresis and then transferred onto a
polyvinylidene difluoride membrane. After blocking, membranes were incubated
with primary anti-ROCK1 (ab134181, Abcam, Cambridge, UK; 1:1,000), anti-cylinD1
(ab251892, Abcam, 1:500), anti-p21 (ab218311, Abcam, 1:1,000), anti-E-cadherin
(ab194982, Abcam, 1:500), and anti-MMP2 (ab97779, 1:1,000) antibodies for
overnight. The following day, secondary anti-rabbit or anti-mouse antibodies
(ab6940, ab97035, Abcam, 1:2,000) were added and incubated with membranes for 1
h at room temperature. The captured bands were quantified using Image Lab™
Software (Bio-Rad, Hercules, California, USA).
lysis buffer and qualified by a bicinchoninic acid assay detecting kit (Thermo
Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were boiled at 100°C in
sodium dodecyl sulfate (SDS) sample buffer for 5 min and separated with
SDS-polyacrylamide gel electrophoresis and then transferred onto a
polyvinylidene difluoride membrane. After blocking, membranes were incubated
with primary anti-ROCK1 (ab134181, Abcam, Cambridge, UK; 1:1,000), anti-cylinD1
(ab251892, Abcam, 1:500), anti-p21 (ab218311, Abcam, 1:1,000), anti-E-cadherin
(ab194982, Abcam, 1:500), and anti-MMP2 (ab97779, 1:1,000) antibodies for
overnight. The following day, secondary anti-rabbit or anti-mouse antibodies
(ab6940, ab97035, Abcam, 1:2,000) were added and incubated with membranes for 1
h at room temperature. The captured bands were quantified using Image Lab™
Software (Bio-Rad, Hercules, California, USA).
5
Immunohistochemical Analysis of Cartilage Markers
6
Western Blot Analysis of Key Signaling Proteins
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After lysing cells in RIPA lysis buffer, total protein samples were acquired, followed by protein separation on 10% SDS-PAGE (Millipore, Bedford, MA, USA) and shifting to PVDF membranes (Millipore). After sealing in 5% nonfat milk, membranes were probed all night at 4 °C with primary antibodies against the internal control GAPDH (ab8245, Abcam) or Tubulin (ab7291, Abcam), as well as MAML2 (ab245612, Abcam), p21 (ab218311, Abcam), HES-1 (ab71559, Abcam), SRSF1 (ab38017, Abcam), ADAR (ab88574, Abcam) and RBPJ (ab25949, Abcam). Following washing in TBST, the HRP-tagged secondary antibody was added for 2 h at room temperature. Finally, ECL luminous liquid (Pierce, Rockford, IL) was added for detection. Experiments were performed in triplicate.
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