Small interfering RNA oligonucleotides specific to EGFR, GSK‐3α, and GSK‐3β were obtained from Santa Cruz Biotechnology. The siRNA negative control was purchased from Thermo Fisher Scientific. Transfection of siRNAs into cells was carried out using Lipofectamine RNAimax (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Gsk 3α
Manufactured by Santa Cruz Biotechnology
Sourced in United States
GSK-3α is a serine/threonine protein kinase that plays a key role in regulating diverse cellular processes. It is involved in the control of glycogen metabolism, gene transcription, and cell fate determination. GSK-3α is a widely expressed enzyme that has been implicated in various pathological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Gsk3β, by BD (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Gsk 3β, by Santa Cruz Biotechnology (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Gapdh, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Hsp60, by Santa Cruz Biotechnology (1 mentions)
Whatman, by Cytiva (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
4 protocols using gsk 3α
1
siRNA-Mediated Gene Silencing Assay
2
Western Blot Analysis of Cellular Proteins
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Cells were rinsed in PBS and lysed in RIPA (radioimmunoprecipitation assay) lysis buffer (19) containing Complete™ Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche) on ice for 15 minutes, cleared by centrifugation and used directly or frozen on dry ice for later use. 20 μg of lysate was resolved on 8% or 10% SDS polyacrylamide gels, transferred to nitrocellulose membranes (Whatman). Nonspecific binding was blocked using 3% BSA/TBST (TBS with 0.05% Tween-20), and blots were probed overnight at 4°C with primary antibodies. Blots were washed in TBST and antigens were detected using HRP-conjugated secondary antibodies as previously described [1 (link), 59 (link)]. Primary antibodies, diluted 1:1000, were to GSK-3α (Santa Cruz Biotechnology sc-5264), GSK-3β (BD Biosciences 610202), HSP60 (Santa Cruz Biotechnology), GAPDH (Sigma), and to NFkB p65 (D14E12), RelB (C1E4), c-Rel, p105/p50 and p100/p52 (18D10) (all Cell Signaling).
3
Histopathological Evaluation of Murine Kidney Injury
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Formalin-fixed mouse kidney were embedded in paraffin and prepared in 3μm-thick sections. Sections were processed for PAS staining. The morphologic features of all the sections were assessed by a single observer in a blinded manner. A semiquantitative glomerular damage index was used to evaluate the degree of glomerular damage [44 (link)]. The severity of injury for each glomerulus was graded from 0 to 5 as follows: 0 represents no lesions; 1, damage of <20% of the glomerulus; and 2, 3, 4, and 5, damage of 20% to 40%, >40% to 60%, and >60% to 80%, and >80% of the glomerulus, respectively. A whole-kidney average glomerular damage index was obtained by averaging scores from all glomeruli on one section [45 (link)–47 (link)]. Immunoperoxidase staining was performed with a Vectastain ABC kit (Vector Laboratories, Burlingame, California, USA) by using of primary antibodies against GSK3α (Santa Cruz Biotechnology, Santa Cruz, CA), GSK3β (Cell Signaling, Danvers, MA), and Nrf2 (Santa Cruz). As a negative control, the primary antibody was replaced by preimmune IgG from the same species and no specific staining was noted.
4
Immunofluorescence Staining of GSK-3 and NF-κB
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Cells were plated on gelatin-coated glass coverslips 48 h before transfection. Cells were fixed for 15 min in 4% PFA, permeabilized with 0.1% Triton for 10 min, incubated for 1 h with blocking buffer (50 mM glycine, 2% BSA, 0.01% Na-azide in PBS) to block nonspecific binding and probed for 2 h with primary antibodies diluted in wash buffer (blocking buffer 1:10 in PBS). Primary antibodies used were to GSK-3α (1:500, Santa Cruz Biotechnology sc-5264), GSK-3β (1:500, BD Biosciences 610202), p65 (1:200, Santa Cruz Biotechnology) and p100/52 (1:250, Abcam). Immune complexes were detected with appropriate Alexa488- or Alexa594-congugated secondary antibodies (Invitrogen) diluted 1:500 in wash buffer and mounted using Vectashield® (Vector Labs), and DAPI (diamidino-2-phenylindole) was used to stain nuclei. Confocal images were acquired as previously described [59 (link)] with an SP2 microscope station (Leica) using a 63 × 1.3 NA lens.
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