Cells were lysed with RIPA buffer, and the protein concentrations were determined by BCA assay (Solarbio, #9c0020, Beijing, China). Total protein (40 μg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel and electrotransferred to polyvinylidene fluoride membranes. The membranes were incubated overnight at 4°C with rabbit monoclonal anti-hnRNP C1+C2 (Abcam, ab133607, Cambridge, England), and anti-LC3A/B, anti-ATG3, anti-ATG5, anti-ATG7, and anti-BECLIN1 (for autophagy detection, Abcam, ab133607, Autophagy Antibody Sampler Kit 4445, England). Alternatively, blots were incubated with rabbit polyclonal anti-14-3-3ε (Abcam, ab43057, Cambridge, England), anti-GAPDH (ABclonal, A10868, Wuhan, China), mouse anti-histone H3 (Beyotime, AF0009, Shanghai, China). Blots were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Later, they were probed using the ECL Reagent and visualized by a Western blotting analysis detection system. Relative quantitative measurement of Western blotting was performed using ImageJ software.
Ab133607
Manufactured by Abcam
Sourced in United Kingdom
Ab133607 is a monoclonal antibody that targets the human CD4 protein. It can be used for a variety of research applications involving the detection and analysis of CD4-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti histone h3, by Beyotime (1 mentions)
Bca assay, by Solarbio (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Ab92355, by Abcam (1 mentions)
Odyssey, by LI COR (1 mentions)
Ab271136, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
4 protocols using ab133607
1
Autophagy Protein Expression Analysis
2
RNA Immunoprecipitation Assay Protocol
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For RNA immunoprecipitation (RIP) assay, a Magna RIP Kit (Millipore, Billerica, MA, USA) was adopted according to the manufacturer’s protocols. The whole cell lysate was incubated in RIP buffer where magnetic beads were absorbed by anti-hnPNRC (ab133607; 1:100; Abcam, Cambridge, MA, USA) or anti-IgG (ab218427; 1:100; CST, Boston, MA, USA). IgG was a normalization control. After the digestion of proteinase K, co-precipitated RNAs including LINC00662 and AK4 were harvested, purified and detected by qRT-PCR.
3
RNA-Protein Binding Assay by EMSA
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For EMSA assays, synthesized RNA labelled with a 5′fluorescent DY781 dye and containing a three base extension was used (5′-DY781-AGUGGCUUAUGCCUGUA/GAUCCCAACAC). Unlabeled RNA was used for competition assays.
Protein-RNA-binding reactions, containing 1 μM labelled RNA and 3 μM ILF3 or hnRNPC in a 10 μl reaction volume, were carried out on ice for 45 min. Competition assays were also carried out in a 10-μl reaction volume. A 1:1 molar ratio complex of RNA and proteins was prepared by incubation on ice for 1 h. The complex at a final concentration of 0.5 μM was mixed with 50 μM unlabeled competitor RNA. Monoclonal antibodies specific for ILF3 (Abcam, ab92355) and hnRNPC (Abcam, ab133607) were added to the binding reaction after complex formation. A 6% polyacrylamide gel in 0.5× Tris/Borate/EDTA buffer (TBE) was pre-run at 4°C for 1 h. Samples were then mixed with 2 μl of native gel loading buffer to a total volume of 6 μl and run on the gel for 2 h. The gel was scanned on a LICOR Odyssey fluorescent infrared scanner at 800 nm. Images were converted to greyscale by ImageJ.
Protein-RNA-binding reactions, containing 1 μM labelled RNA and 3 μM ILF3 or hnRNPC in a 10 μl reaction volume, were carried out on ice for 45 min. Competition assays were also carried out in a 10-μl reaction volume. A 1:1 molar ratio complex of RNA and proteins was prepared by incubation on ice for 1 h. The complex at a final concentration of 0.5 μM was mixed with 50 μM unlabeled competitor RNA. Monoclonal antibodies specific for ILF3 (Abcam, ab92355) and hnRNPC (Abcam, ab133607) were added to the binding reaction after complex formation. A 6% polyacrylamide gel in 0.5× Tris/Borate/EDTA buffer (TBE) was pre-run at 4°C for 1 h. Samples were then mixed with 2 μl of native gel loading buffer to a total volume of 6 μl and run on the gel for 2 h. The gel was scanned on a LICOR Odyssey fluorescent infrared scanner at 800 nm. Images were converted to greyscale by ImageJ.
4
Western Blot Analysis of Protein Expression
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After trypsin digestion, cells from different groups were collected and lysed using RIPA buffer (enhanced version, 89,900, Invitrogen, Car, Cal, USA) containing protease inhibitors. The protein concentration was quantified using a BCA protein assay kit (23,235, Invitrogen, Car, Cal, USA). Subsequently, protein samples were separated by 10% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes. After blocking with 5% BSA for 2 h at room temperature to block non-specific binding, diluted primary antibodies, including DDR1 (CST, 1:1000, #3917), GAPDH (CST, 1:1000, #92,310), VIRMA (Abcam, 1:1000, ab271136), and HNRNPC (Abcam, 1:1000, ab133607), were added and incubated overnight at 4 °C. After washing, the membranes were incubated with HRP-labeled goat anti-rabbit secondary antibodies at room temperature for 1 h. ECL working solution (35,055, Invitrogen, Car, Cal, USA) was then used for membrane exposure for 1 min, and excess ECL reagent was removed. Finally, Western blot images were exposed and analyzed using the Bio-Rad Gel Imaging system and ImageLab software. GAPDH was used as an internal reference, and each experiment was repeated three times.
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