Western blot was performed as described previously (21 (link)). LV tissue was homogenized in radioimmunoprecipitation buffer containing 1% protease inhibitor cocktail, and the concentration of the supernatant was measured by means of BCA. After the target proteins were loaded onto an SDS polyacrylamide gel, proteins were transferred from the gel to a PVDF membrane. Subsequently, the membranes were incubated overnight with Areg, mTOR (A2445, ABclonal), AKT (A18120, ABclonal), P-AKT (AP0655, ABclonal), or P-mTOR (AP0094, ABclonal) antibody after being blocked for 1 h. After washed in TBST, the membranes were incubated with HRP-linked secondary antibody for 60 min at room temperature. The protein bands were detected using an enhanced chemiluminescence agent (ECL reagents) and analyzed.
Ap0094
Manufactured by ABclonal
Sourced in China
AP0094 is a laboratory equipment product offered by ABclonal. It is designed for a specific core function, but a detailed and unbiased description cannot be provided while maintaining the requested constraints.
Automatically generated - may contain errors
Lab products found in correlation
A2445, by ABclonal (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence, by Cell Signaling Technology (1 mentions)
A6058, by ABclonal (1 mentions)
A0674, by ABclonal (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab109012, by Abcam (1 mentions)
Ab191606, by Abcam (1 mentions)
Bs 0295g hrp, by Bioss Antibodies (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab45688, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab270993, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
4 protocols using ap0094
1
Western Blot Analysis of Key Signaling Proteins
2
Western Blot Analysis of Liver Proteins
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Based on the method described in our laboratory [31 (link)], we used the western blot to detect mTOR, p-mTOR, S6, p-S6, Nrf2, and Keap1 protein expression levels. Briefly, the liver was prepared with the RIPA buffer (Thermo Fisher Scientific). Then, the proteins (40 μg from each sample) were separated on 8% or 12% SDS–polyacrylamide gel, depending on the molecular weight of proteins. They were transferred to the PVDF membranes (Thermo Fisher Scientific), blocked with 8% (w/v) skimmed milk in the TBST buffer (20 mM Tris–HCl, 0.1% Tween 20, 150 mM sodium chloride, pH 7.5) for 1 h, washed thrice with the TBST buffer for 10 min each, and followed by the incubation with specific primary antibodies, such as rabbit anti-p-mTOR-S2448 (1 : 2000, AP0094; Abclonal, Wuhan, China), anti-mTOR (1 : 1000, A2445; Abclonal), anti-p-S6- S235/236 (1 : 1000, AP0538; Abclonal), anti-S6 (1 : 1000, A6058; Abclonal), anti-Nrf2 (1 : 1000, A0674; Abclonal), and anti-Keap1 (1 : 5000, A17061; Abclonal) for overnight at 4°C. They were then incubated with goat anti-rabbit secondary antibody (1 : 10000). Immunoreactive bands were visualized via the enhanced chemiluminescence (Cell Signaling Technology) and quantified via the densitometry using ImageJ software (version 1.42, NIH).
3
Western Blot Analysis of Autophagy and EMT Markers
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Proteins of lung tissues and RLE‐6TN cells were extracted with RIPA Lysis Buffer (Beyotime, Shanghai, China). After quantification, the protein samples were subjected to SDS-PAGE, then blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies LC3B (ab48394, 1 µg/ml, Abcam), Beclin-1 (ab207612, 1:2000, Abcam), p62 (ab109012, 1:10000, Abcam), E-cadherin (ab231303, 1 µg/ml, Abcam), N-cadherin (ab76011, 1:5000, Abcam), Vimentin (ab92547, 1:1000, Abcam), α-SMA (ab124964, 1:10000, Abcam), collagen I (ab270993, 1:1000, Abcam), Fibronectin 1 (ab45688, 1:1000, Abcam), p-PI3K (ab182651, 1:1000, Abcam), PI3K (ab191606, 1:1000, Abcam), p-Akt (ab38449, 1:500, Abcam), Akt (ab38449, 1:500, Abcam), p-mTOR (AP0094, 1:500, ABclonal), mTOR (A2445, 1:500, ABclonal), CDC27 (A3333, 1:1000, ABclonal), and GAPDH (ab8245, 1:2000, Abcam) at 4°C overnight, followed by incubation with secondary antibody (bs-0295 G-HRP, 1:1000, Bioss). ECL chemiluminescence (Beyotime) was applied for analysis.
4
Protein Expression Analysis in Kidney Tissue
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Proteins were isolated from kidney tissue and MPC-5, and protein levels were assayed. Briefly, protein extracts were boiled in RIPA buffer (Beyotime, Shanghai, China) and separated by SDS-PAGE electrophoresis, transferred to NC membranes, closed, and then incubated overnight at 4°C with the primary antibodies nephrin (NPHS1 A3048; ABclonal, China), podocin (NPHS2 20384-1-AP; Proteintech, China), and synaptopodin (A8484; ABclonal, China) to detect their protein levels in kidney tissue and MPC-5. Beclin1 (A17028; ABclonal, China), p62 (A11250; ABclonal, China), LC3B (A19665; ABclonal, USA), Phospho-mTOR-S2448 (AP0094, ABclonal, China), mTOR (AF6308, Affinity, China), Phospho-ULK1-S757 (AP0736, ABclonal, China) and ULK1 (AF4687, Affinity, China) antibodies were assessed to detect their protein levels in MPC-5. GAPDH (AC002; Abclonal, China) or Tubulin (A17913; Abclonal, China) were used as reference proteins. The blots were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and visualized using the Bio-Rad Gel Doc EZ imaging system (Bio-Rad Laboratories, Hercules, CA, USA) and enhanced chemiluminescence reagents (Millipore, Boston, MA, USA). All western blot analyses were performed ≥3 times and protein bands were quantified using the ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).
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