Whole left ventricles previously stored in −80 °C were homogenized in Protein Extraction Reagent Type 4 (Sigma C0356), supplemented with protease inhibitor (ThermoFisher A32953) for detection of several proteins by Western blotting. Primary antibodies utilized were SERCA2 ATPase (ThermoFisher, cat. #MA3–919), GRP78/ HSPA5 (ThermoFisher, cat. #PA1–16857), Beclin1 (Thermofisher, cat. #PA1–16857), ChChd3 (Thermofisher, cat. #PA 5–31578), Mfn1 (ThermoFisher, cat. #PA5–38042), OPA1 (ThermoFisher, cat. #PA1–16991), Fis1 (PA 1–41082), Drp1 (PA5–34768), EphrinA1 (SantaCruz Biotechnology, cat #Sc-911), GAPDH (Cell Signaling, cat #2118), alpha tubulin (Invitrogen, cat. #138000), and phospho-alpha tubulin (Tyr272) (ThermoFisher, cat. #PA5–37831). Membranes were blotted using either mouse IgG-Fc secondary antibody (Thermo scientific cat. #31455) or rabbit IgG HRP-conjugated antibody (R&D systems cat. #HAF008), and the chemiluminescent substrate SuperSignal West Pico PLUS (ThermoFisher, cat. #34078), and imaged in a ChemiDoc-ItTS2 810 Imager, UVP.
Grp78 hspa5
Manufactured by Thermo Fisher Scientific
GRP78/HSPA5 is a molecular chaperone protein that plays a crucial role in the folding and maturation of proteins within the endoplasmic reticulum (ER). It is also known as the 78 kDa glucose-regulated protein (GRP78) or the heat shock protein family A (Hsp70) member 5 (HSPA5). GRP78/HSPA5 assists in the proper folding and assembly of proteins, helps prevent the aggregation of misfolded proteins, and facilitates the transport of proteins across the ER membrane.
Automatically generated - may contain errors
Lab products found in correlation
Ephrina1, by Santa Cruz Biotechnology (1 mentions)
Alpha tubulin, by Thermo Fisher Scientific (1 mentions)
Protein extraction reagent type 4, by Merck Group (1 mentions)
Beclin1, by Thermo Fisher Scientific (1 mentions)
Rabbit igg hrp conjugated antibody, by R&D Systems (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Bio-Rad (1 mentions)
Caspase 1, by Adipogen (1 mentions)
2 protocols using grp78 hspa5
1
Cardiac Protein Expression Analysis
2
Western Blot Analysis of Stress Proteins
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Protein lysates from KerCT cells and skin tissues were collected in ice-cold RIPA lysis buffer (Bio-Rad) to perform western blots analysis. Using a Bio-Rad protein assay DC kit (Hercules, CA), protein concentration was measured in these samples. 30-40 µg protein from these samples was electrophoretically separated on SDS-PAGE and transferred to PVDF membrane.
Following protein transfer, nonspecific sites were blocked by 5% nonfat dry milk for 1h. Milkblocked membranes were probed with primary antibodies p-HSP90α (CST, 3488), HSP90 (CST, 8165), Cleaved caspase-3 (CST, 9661), p-P38 (CST, 4511), NLRP3 (CST, 15101), Caspase-1 (AdipoGen, AG-20B-0042-C100), GRP78/HSPa5 (Thermo Fisher, PA5-34941), HSP70/HSPa1a) (CST,4872), HSP27 (HSPb1) (Thermo Fisher, MA5-32473) overnight by incubation at 4°C. Next day, the membrane was washed with TBST buffer for 3 times of 10 minutes each. Finally, the membranes were incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized with an iBright1000 imaging system (ThermoFisher, Waltham, MA) with the help of enhanced chemiluminescence according to the manufacturer's instructions (Santa Cruz Biotechnology, Dallas, TX, USA). Βactin (3700, CST) was used as an endogenous control. Densitometry analysis of band intensity was performed using ImageJ software.
Following protein transfer, nonspecific sites were blocked by 5% nonfat dry milk for 1h. Milkblocked membranes were probed with primary antibodies p-HSP90α (CST, 3488), HSP90 (CST, 8165), Cleaved caspase-3 (CST, 9661), p-P38 (CST, 4511), NLRP3 (CST, 15101), Caspase-1 (AdipoGen, AG-20B-0042-C100), GRP78/HSPa5 (Thermo Fisher, PA5-34941), HSP70/HSPa1a) (CST,4872), HSP27 (HSPb1) (Thermo Fisher, MA5-32473) overnight by incubation at 4°C. Next day, the membrane was washed with TBST buffer for 3 times of 10 minutes each. Finally, the membranes were incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized with an iBright1000 imaging system (ThermoFisher, Waltham, MA) with the help of enhanced chemiluminescence according to the manufacturer's instructions (Santa Cruz Biotechnology, Dallas, TX, USA). Βactin (3700, CST) was used as an endogenous control. Densitometry analysis of band intensity was performed using ImageJ software.
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