HCC827OR and PC9OR cells were treated with FYN at IC50 for 48 h to determine protein expression levels. The cells were then lysed in ice-cold RIPA buffer (Beyotime Inc., China, P0013B), and a BCA Protein Assay Kit (Beyotime Inc., China, P0010) was used to quantify protein concentrations. Total proteins in different groups were subjected to SDS-PAGE gel separation, then transferred to polyvinylidene difluoride membranes (Merck Millipore). After blocking with 5% skim milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. Secondary antibodies were then added, and the preparations were incubated for 2 h. The probed membranes were developed with ECL solution (Sangon, Shanghai, China, C510043-0100). Primary antibodies against SRSF1 (Cat# 14908), GSK3B (Cat# 5558), β-catenin (Cat# 8480), and GAPDH (Cat# 5174) were acquired from Cell Signaling Technology. The primary antibody against PCNA (Cat# sc-56) was acquired from Santa Cruz Biotechnology.
Primary antibody against pcna
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The primary antibody against PCNA (Proliferating Cell Nuclear Antigen) is a laboratory tool used to detect the presence and distribution of PCNA, a protein involved in cell proliferation. This antibody can be used in various immunoassay techniques to identify and quantify PCNA expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Molecular imager chemidoc xrs system, by Bio-Rad (3 mentions)
P egfr y1068, by Abcam (3 mentions)
Primary antibodies against s326 p hsf1, by Bioss Antibodies (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gsk3b, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ecl solution, by Sangon (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Chemiluminescent detection system, by Santa Cruz Biotechnology (1 mentions)
Quantity one imaging software, by Bio-Rad (1 mentions)
Peroxidase conjugated anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Chemicdoc xrs system, by Bio-Rad (1 mentions)
5 protocols using primary antibody against pcna
1
Quantifying Protein Expression in HCC827OR and PC9OR Cells
2
Investigating IL-4 Effects on NSC Proliferation
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To confirm the effect of IL-4 on proliferation of NSCs and to investigate its the mechanism, the expression of proliferating cell nuclear antigen (PCNA) using western blot analysis. Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w/v) Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 10 μg/ml aprotinin. Cell lysates were separated by 12% SDSpolyacrylamide gel electrophoresis, and electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with blocking buffer (1× Tris-buffered saline, 1% BSA, 1% nonfat dry milk) for 1 h and then incubated with the primary antibody against PCNA (Santa Cruz Biotechnology; 1:1,000) for overnight at 4°C. Primary antibodies were visualized with peroxidase-conjugated anti-rabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology, Inc., USA). The ChemicDoc XRS system (Bio-Rad) was used for visualization and quantification using Quantity One imaging software (Bio-Rad).
3
Quantitative Western Blot Analysis
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Western blot experiments were performed as described previously [13 ]. Briefly, total protein from cells was prepared according to the manufacturer’s instructions. After protein concentration determination and gel electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes, and then the membranes were blocked in 10% nonfat dry milk powder (dissolved in PBS containing 0.1% Tween-20, PBST) for 2 h and subsequently incubated with primary antibodies overnight at 4 °C. The primary antibodies against HSP70 and β-actin that were used in this study were from Proteintech Group. The primary antibodies against S326 p-HSF1 was obtained from Bioss, the primary antibodies against HSF1, Amylase, CK19, EGFR and Y1068 p-EGFR were purchased from Abcam. The primary antibody against PCNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Following incubation with secondary HRP-coupled antibodies for 1 h at room temperature, the membranes were washed with PBST, and the immunocomplexes were detected using an enhanced chemiluminescence kit and a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories). β-actin was used as an internal control.
4
Western Blot Technique: Protein Detection
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Western blot experiments were performed as described previously [13] . Brie y, total protein from cells was prepared according to the manufacturer's instructions. After protein concentration determination and gel electrophoresis, the proteins were transferred onto polyvinylidene di uoride membranes, and then the membranes were blocked in 10% nonfat dry milk powder (dissolved in PBS containing 0.1% Tween-20, PBST) for 2 h and subsequently incubated with primary antibodies overnight at 4 °C. The primary antibodies against HSP70 and β-actin that were used in this study were from Proteintech Group. The primary antibodies against S326 p-HSF1 was obtained from Bioss, the primary antibodies against HSF1, Amylase, CK19, EGFR and Y1068 p-EGFR were purchased from Abcam. The primary antibody against PCNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Following incubation with secondary HRP-coupled antibodies for 1 h at room temperature, the membranes were washed with PBST, and the immunocomplexes were detected using an enhanced chemiluminescence kit and a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories). β-actin was used as an internal control.
5
Western Blot Analysis of Cellular Proteins
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Western blot experiments were performed as described previously [13] . Brie y, total protein from cells was prepared according to the manufacturer's instructions. After protein concentration determination and gel electrophoresis, the proteins were transferred onto polyvinylidene di uoride membranes, and then the membranes were blocked in 10% nonfat dry milk powder (dissolved in PBS containing 0.1% Tween-20, PBST) for 2 h and subsequently incubated with primary antibodies overnight at 4 °C. The primary antibodies against HSF1, HSP70 and β-actin that were used in this study were from Proteintech Group. The primary antibodies against amylase, CK19, EGFR, p-EGFR (Y1068) were purchased from Abcam. The primary antibody against PCNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Following incubation with secondary HRP-coupled antibodies for 1 h at room temperature, the membranes were washed with PBST, and the immunocomplexes were detected using an enhanced chemiluminescence kit and a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories). β-actin was used as an internal control.
Following incubation with secondary HRP-coupled antibodies for 1 h at room temperature, the membranes were washed with PBST, and the immunocomplexes were detected using an enhanced chemiluminescence kit and a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories). β-actin was used as an internal control.
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