Renal tissues were lysed in cell lysates containing the protease inhibitor cocktail (Beyotime, P1005). Equal amounts of the prepared protein were resolved using SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% milk for 1 h, the membranes were blocked with the following primary antibody diluted 1:1,000 (prepared with PBS) at 4°C overnight: Collagen I (Zen-bio, 343,277), Collagen Ⅲ (Zen-bio, 250,064), p-PERK (Affinity, DF7576), p-IRE1α (Affinity, DF8322), GRP78 (Proteintech, 11587-1-AP), ATF-6 (Proteintech, 24169-1-AP), CHOP (Cell Signaling Technology, 2895S), TNF-α (Proteintech, 17590-1-AP), IL-6 (Santa Cruz Biotechnology, sc-57315), IL-10 (Proteintech, 20850-1-AP), VEGF (Proteintech, 15204-1-AP), ACE (Proteintech, 21115-1-AP), AT1 (Abcam, EPR3873), and β-actin (Solarbio, K200058M). The membranes were washed with PBST for three times and then treated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Beyotime, A0216 or A0208) for 2 h at room temperature. Signals were visualized using the ChemiDocXRS + Imaging System (Bio-Rad). All experiments were performed in triplicate and tissue samples were prepared independently for each experiment. The densitometric values of the bands were obtained using Image J software (NIH, Bethesda, MD) and subjected to statistical analysis.
P perk
Manufactured by Affinity Biosciences
Sourced in United Kingdom
The P-PERK is a laboratory instrument designed for the detection and quantification of phosphorylated PERK (Protein Kinase R-like Endoplasmic Reticulum Kinase), a key marker of the unfolded protein response in cells. The device utilizes advanced detection technologies to provide accurate and reliable measurements of P-PERK levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
P ire1α, by Affinity Biosciences (2 mentions)
P eif2α, by Affinity Biosciences (2 mentions)
Ire1α, by Affinity Biosciences (2 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (1 mentions)
Grp78, by Proteintech (1 mentions)
Il 10, by Proteintech (1 mentions)
β actin, by Solarbio (1 mentions)
Interleukin 6 (il 6), by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Tnf α, by Proteintech (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
2 3 5 triphenyltetrazolium chloride ttc, by Merck Group (1 mentions)
Caspase 3, by Affinity Biosciences (1 mentions)
Caspase12, by Affinity Biosciences (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
Puromycin, by Kerafast (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
P erk, by Abcam (1 mentions)
5 protocols using p perk
1
Renal Fibrosis Protein Analysis
2
Neuroprotective Effects of Edaravone
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Edaravone injection was purchased from Sinopharm Group Guorui Pharmaceutical Co. LTD (China). Sul-F (> 95% pure) was bought from Shijiazhuang Hairui Pharmaceutical Technology Co. LTD (China). 2,3,5-triphenylte-trazolium chloride (TTC) was acquired from Sigma (USA). Hematoxylin-eosin staining (HE) kit and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) kit were purchased from Biyuntian Biotechnology Co. LTD (China). Bcl-2, Caspase3, Bax, CHOP, p-PERK, p-eIF2α, p-IRE1, Caspase12 and ATF4 primary antibodies were purchased from Affinity (China).
3
Western Blot Analysis of UPR Markers
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According to a protocol described previously, 20 μg protein of cell lysates were Western imprinted (Wang et al., 2016 (link)) and the membrane was co-incubated with the following antibodies: β-Actin (Abcam, #AC004, UK), eIF2α (Cell Signaling Technology, #ab242148, USA), P-eIF2α (Ser51, Cell Signaling Technology, #ab32157, USA) GRP78 (Cell Signaling Technology, #3177S, USA), PERK (Abcam,ab229912,UK), p-PERK(Affinity, DF-7576), LC3B (Abcam, ab192890, UK), Puromycin (Kerafast, EQ0001,USA). The band density was measured (Quantity One software) and normalized to β-Actin.
4
Protein Expression Analysis in HCT116 Cells
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Total protein was extracted from HCT116 cells in RIPA buffer. A BCA kit was applied to measure the protein concentration. Then, proteins were electrophoresed on SDS-PAGE gels and transferred onto PVDF membranes. After non-specific blocking with BSA for 1 h, the membranes were probed with primary antibodies. Then, the membrane was incubated with secondary antibodies at room temperature for 1 h. The signals were captured through ECL Blotting Detection Reagents. The following primary antibodies were used in this study: Bip (CST#3177); IRE1α (CST#3294); PERK (CST#5683); p-PERK (Affinity#DF7576); p-eIF2α (CST#3398); eIF2α (CST#5324); ATF4 (CST#11815); Aurora A (CST#14475); and GAPDH (CST#2118).
5
Western Blot Analysis of ER Stress Markers
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Cells were lysed by RIPA lysis buffer (KeyGEN BioTECH), centrifuged for 15 min at 12,000 g, and determined by BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Proteins were separated by 8–12% SDS/PAGE and transferred to polyvinylidene difluoride membranes. The membrane was blocked with TBS-T (containing 5% bovine serum albumin) for 1 h prior at 25 °C and western blotting with polyclonal antibody at 4 °C overnight. The primary antibodies included rabbit polyclonal antibody against GRP78, PKC-δ, p-PKC-δ, CHOP, CASPASE12 (1:1000, Abcam, UK); SREBP1c, FOXO1, SERCA2, CANX, IP3R, p-PERK, PI3Kp85, AKT, p-AKT, IRE1-α, p-IRE1-α (1:1000, Affinity, USA); PERK, ATF6, ATF4 (1:1000, Cell Signaling Technology, USA); GAPDH (1:5000, Proteintech Group, Inc., USA). After 1 h incubation with corresponding secondary antibody, the immunoreactive bands were visualized by a chemiluminescent substrate (ECL, Amersham, USA).
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