WB was conducted as previously reported34 (link). In brief, cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, China) containing 1% phenylmethylsulfonyl fluoride. Extracted protein concentration was measured by the bicinchoninic acid method and stored at −80 °C. A 25-µg protein sample was run on an 8–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad, USA). Afterward, the membrane was blocked in 5% nonfat milk (BD, USA) and incubated with the primary antibodies at 4 °C overnight. Finally, the membrane was incubated with the secondary antibodies and developed under the gel electrophoresis imager (Bio-Rad, USA). The primary antibodies of LC3 (ab192890), P62 (ab207305), and ATG5 (ab109490) were purchased from Abcam, while p53 (10442-1-AP), Zeb1 (21544-1-AP), and GAPDH (10494-1-AP) antibodies were purchased from Proteintech. The secondary antibody anti-Rabbit (SA00001-2) was also purchased from Proteintech.
Ab109490
Manufactured by Abcam
Sourced in United States
Ab109490 is a lab equipment product manufactured by Abcam. It is a core component used in scientific research and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Ab192890, by Abcam (3 mentions)
Ab109268, by Abcam (2 mentions)
Gel electrophoresis imager, by Bio-Rad (1 mentions)
Ab207305, by Abcam (1 mentions)
Nonfat milk, by BD (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Gapdh 10494 1 ap, by Proteintech (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab52472, by Abcam (1 mentions)
Ab40776, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab62557, by Abcam (1 mentions)
Ab203207, by Abcam (1 mentions)
Ab115777, by Abcam (1 mentions)
Ab76149, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Ab5831, by Abcam (1 mentions)
Ab184993, by Abcam (1 mentions)
Ab138492, by Abcam (1 mentions)
Ab210498, by Abcam (1 mentions)
7 protocols using ab109490
1
Western Blot Analysis of Autophagy Markers
2
Autophagy Pathway Protein Expression
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The expressions of ALKBH5, ATG7 (Autophagy related 7), ATG5(Autophagy related 5), Beclin1, ULK1 (Serine/Threonine-Protein Kinase ULK1), PI3KC3, p-MTOR (Mammalian target of rapamycin), and Actin proteins were analyzed by WB. The primary antibodies included monoclonal rabbit anti-ALKBH5 (Abcam, ab234528), rabbit monoclonal to ATG7 (Abcam, ab52472), rabbit monoclonal to ATG5 (Abcam, ab109490), rabbit polyclonal to Beclin1 (Abcam, ab62557), rabbit polyclonal to ULK1 (Abcam, ab203207), rabbit monoclonal to PI3KC3 (Abcam, ab40776), rabbit monoclonal to p-mTOR (Abcam, ab109268). Monoclonal mouse anti-β-actin (Abcam, ab8226) was used as an internal control to evaluate the band density on a gel imaging system.
3
Western Blot Analysis of Autophagy and Cell Cycle Proteins
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The expressions of LC3, ATG7, ATG5, YBX1, P21 and actin proteins were analyzed by western blot18 (link),19 (link). The primary antibodies used include polyclonal rabbit anti- LC3 (Cell Signaling Technology, 4108); rabbit monoclonal to ATG5 (Abcam, ab109490); rabbit monoclonal to ATG7(Abcam, ab183188); rabbit monoclonal to YBX1 (Abcam, ab76149); rabbit monoclonal to P21 (Abcam, ab109520); and monoclonal rabbit anti-beta actin (Abcam, ab115777). The band density was analyzed using a gel imaging system and compared with an internal control.
4
Western Blot Analysis of Autophagy and Fibrosis Markers
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Westrern blot assay was performed as previously described elsewhere [24 (link)]. Briefly, total proteins quantification was done through the BCA method (Beyotime, Shanghai, China). Subsequently, proteins (30 μg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by a transfer into a membrane of polyvinylidene difluoride (PVDF) (Thermo Fisher Scientific). The membrane was later blocked for 1 hour using 5% skimmed milk in TBST, incubated using rabbit anti-human ATG5 (ab109490, 1:1000, Abcam), Beclin-1 (ab210498, 1:1000), LC3I/II (ab192890, 1:1000), p62 (ab109012, 1:1000), collagen I (ab138492, 1:1000), collagen III (ab184993, 1:1000), α-SMA (ab5831, 1:1000), at 4°C overnight. The membrane was subsequently incubated for 1 hr at room temperature using secondary antibody horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000, Solabio). Finally, the band detection was done using the ECL Chemiluminescent Substrate Kit (Thermo Fisher Scientific). β-actin (4970S, 1:1000, Cell Signaling Technology) was as the loading control. Each experiment was performed in triplicate.
5
Autophagy Inhibitors and Platinum-based Chemotherapy
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3-Methyladenine (3-MA) (S2767), Rapamycin (S1039), and Wortmannin (S2758) were purchased from Selleckchem (Houston, TX, USA). Chloroquine diphosphate salt (C6628) was purchased from Sigma-Aldrich (MO, USA). cis-Diamminedichloroplatinum (Cisplatin, DDP, Lot.No:8L012A88) was purchased from QILU Pharmaceutical (Shandong, China). Antibodies against ATG5 (ab109490), ATG7 (ab133528), MITF (ab140606), and LC3B (ab192890) were purchased from Abcam (Cambridge, UK). Antibodies against p62 (18420-1-AP) and GAPDH (60004-1-Ig) were purchased from Proteintech (Chicago, IL, USA). Antibody against LC3B (83506S) and Beclin 1 were purchased from Cell Signaling Technology (Danvers, MA, USA). LAMP-1 (sc-19992) and LAMP-2 (sc-18822) were purchased from Santa Cruz (Dallas, TX, USA).
6
Protein Expression Analysis in Kidney and HUVEC
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Kidney tissues and HUVECs were collected, and the protein concentration was determined by radioimmunoprecipitation analysis (RIPA), lysis buffer solution, and bicinchoninic acid (BCA) method. Proteins were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The primary antibody was then incubated at 4°C overnight. The antibodies included anti-S6K (ab32529, 1:5000, Abcam, UK), anti-p-S6K (ab32529, 1:5000, Abcam, UK), anti-Beclin1 (11306-1-AP, 1: 5000, Proteintech, USA), anti-ATG5 (ab109490, 1:5000, Abcam, UK), anti-LC3 (14600-1-AP, 1: 1000, Proteintech, USA), anti-p62 (18420-1-AP, 1:2000, Proteintech, USA), anti-KLF4 (11880-1-AP, 1: 500, Proteintech, USA), anti-caspase 3 (19677-1-AP, 1:500, Proteintech, USA), anti-p-mTOR (ab109268, 1: 5000, Abcam, Cambridge, UK), anti-mTOR (ab32028, 1:2000, Abcam, UK), and anti-b-actin (60008-1-IG, 1:5000, Proteintech, USA). It was then combined with secondary anti-IgG antibodies (1:5000, SA00001-1; 1:6000, SA00001-2, Proteintech, USA). Visualization and imaging analyses were performed using chemiluminescence (Millipore, USA) and imaging software (GE Healthcare, Life Sciences, USA).
7
Iron Detection and Western Blotting for HL-1 Cells
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The pretreatment of iron-containing DMEM cultured HL-1 cells were described as the foregoing method.
Iron was detected by the Iron Colorimetric Assay Kit (E1042, Beijing applygen, China) according to the manufacturer's instructions. The absorbance was determined by 550nm, which drew the standard curve and calculated the concentration of iron. Western blot analysis Brie y, after collection of the supernatants of the tissue or cell lysates, protein samples (20-25 mg) were separated by sodiumdodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di uoride (PVDF) membranes. The membranes were probed with primary antibodies overnight. Diluted secondary antibodies were used to detect the matching primary antibodies. Further analysis was carried out using chemiluminescence imaging analysis system (Shanghai Clinx Science Instruments Co, China) to quantify the protein bands.
The following blotting reagents and antibodies were used: Prepare cell lysate (RIPA cracking solution, Thermo, USA), the concentrated glue and separation glue (Thermo, USA), anti-GAPDH (60004-1-Lg, Proteintech, USA), anti-LRP6 (sc-25317, Santa Cruz Biotechnology, USA), Goat anti-Mouse IgG HRP (ab205719 or ab6721, abcam, Britian), anti-LC3-A/B (12741T, CST, USA), ATG5 (ab109490, abcam, Britain), P62 (5114T, CST, USA), High sensitive ECL luminescence kit (Thermo, USA).
Iron was detected by the Iron Colorimetric Assay Kit (E1042, Beijing applygen, China) according to the manufacturer's instructions. The absorbance was determined by 550nm, which drew the standard curve and calculated the concentration of iron. Western blot analysis Brie y, after collection of the supernatants of the tissue or cell lysates, protein samples (20-25 mg) were separated by sodiumdodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di uoride (PVDF) membranes. The membranes were probed with primary antibodies overnight. Diluted secondary antibodies were used to detect the matching primary antibodies. Further analysis was carried out using chemiluminescence imaging analysis system (Shanghai Clinx Science Instruments Co, China) to quantify the protein bands.
The following blotting reagents and antibodies were used: Prepare cell lysate (RIPA cracking solution, Thermo, USA), the concentrated glue and separation glue (Thermo, USA), anti-GAPDH (60004-1-Lg, Proteintech, USA), anti-LRP6 (sc-25317, Santa Cruz Biotechnology, USA), Goat anti-Mouse IgG HRP (ab205719 or ab6721, abcam, Britian), anti-LC3-A/B (12741T, CST, USA), ATG5 (ab109490, abcam, Britain), P62 (5114T, CST, USA), High sensitive ECL luminescence kit (Thermo, USA).
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