Paraffin sections (4 mm thick) were deparaffinized and rehydrated in 3% hydrogen peroxide for 15 min at room temperature. Antigen retrieval was performed by heating the sections in citrate buffer at 95°C for 1 h. After blocking endogenous peroxidase with 3% hydrogen peroxide, tissue sections were incubated overnight at 4°C with the primary antibodies against CUL4A (1:100; Abcam, Cambridge, MA, USA) and NF-κB (1:50; Cell Signaling, Danvers, MS, USA). After washing three times with phosphate buffer saline (PBS), a biotinylated secondary antibody (Zhongshan Bio-Tech, Beijing, People’s Republic of China) was added (1:100) and incubated for 10 min at room temperature. Tissue sections were developed with 3,3′-diaminobenzidine and counterstained with hematoxylin. The rate of staining intensity was scored as 0 (no staining), 1 (light yellow), 2 (yellow brown), and 3 (brownish yellow staining). The positively stained tumor cells were graded as 0 (no positively stained cells), 1 (<10% of positive cells), 2 (10–50% of positive cells), and 3 (>50% of positive cells). The immunostaining index was calculated as the rate of positively stained tumor cells multiplied by the staining intensity score; tumors with indexes 0–4 were considered negative, and those with indexes 5–9 were considered positive.
Cul4a
Manufactured by Abcam
Sourced in United States, United Kingdom
Cul4A is a protein complex component that is involved in ubiquitin-mediated proteolysis. It acts as a substrate recognition component of the Cul4-DDB1 E3 ubiquitin-protein ligase complex, which mediates the ubiquitination and subsequent proteasomal degradation of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Tubulin antibody, by Merck Group (1 mentions)
Fibronectin, by Cell Signaling Technology (1 mentions)
Lats1, by Cell Signaling Technology (1 mentions)
P yap, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti ha beads, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ecl blotting analysis system, by GE Healthcare (1 mentions)
Anxa10, by GeneTex (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Bioworld Technology (1 mentions)
8 protocols using cul4a
1
Immunohistochemical Analysis of CUL4A and NF-κB
2
Purification and Characterization of DNA Repair Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell extracts (WCE) were prepared as previously described (26 (link),27 (link)). WCE from 20 g HeLa cell pellets (Cilbiotech, Belgium) was fractionated using column chromatography, and proteins present in active fractions from the final Mono-Q chromatography were identified by tandem mass spectrometry, as recently described (27 (link),28 (link)). Following each chromatography stage, protein fractions were analysed for in vitro BER activity using a free or mononucleosome substrate and active fractions pooled for the next chromatography step. Immunoblotting was performed as described in the references above, using the Odyssey Image Analysis System for protein detection and quantification. Primary antibodies raised against APE1 were kindly provided by Prof. G.Dianov, HECTD1 and Mule antibodies were from Bethyl Laboratories (Montgomery, USA), tubulin antibodies were from Sigma (Dorset, UK), Cul4A, DDB1 and histone H2B and H4 antibodies were from Abcam (Cambridge, UK), and histone H3 and H4 antibodies were from Cell Signaling (London, UK).
3
Investigating CUL4A-LATS1 Interactions in Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from GC tissues and cells by lysing in ice-cold lysis buffer. The proteins were electrophoresed on an SDS-PAGE gel, transferred to a PVDF membrane (Millipore, Danvers, MA, USA), and probed with a primary antibody targeted against CUL4A (Abcam, Cambridge, MA, USA), p21 (Abcam), p27 (Abcam), E-cadherin (Abcam), N-cadherin (Abcam), MST1 (Cell Signaling, Danvers, MA, USA), MST2 (Cell Signaling), LATS1 (Cell Signaling), YAP (Cell Signaling), p-YAP (Cell Signaling), vimentin (Cell Signaling), fibronectin (Cell Signaling) or β-actin (Cell Signaling). After incubating with the primary antibody, membranes were washed with TBS/0.05% Tween-20 (TBST) and incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) for 1 h at room temperature. After washing 3 times with (TBST) for 15 min, the membranes were developed using an ECL plus western blotting detection system.
For IP assays, HGC-27 cells were transfected with the Flag-LATS1 plasmid or co-transfected with Flag-LATS1 and HA-CUL4A plasmids, then treated with MG132 (10 μmoL) for 6 h, and lysed with the lysis buffer. Cell lysates were incubated with 5 μL of anti-HA beads (Catalog Number E6779, Sigma, USA) at 4°C for 4 h, then centrifuged and washed with RIPA buffer for 3 times. IP samples were immunoblotted in subsequent experiments.
For IP assays, HGC-27 cells were transfected with the Flag-LATS1 plasmid or co-transfected with Flag-LATS1 and HA-CUL4A plasmids, then treated with MG132 (10 μmoL) for 6 h, and lysed with the lysis buffer. Cell lysates were incubated with 5 μL of anti-HA beads (Catalog Number E6779, Sigma, USA) at 4°C for 4 h, then centrifuged and washed with RIPA buffer for 3 times. IP samples were immunoblotted in subsequent experiments.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole protein was extracted using mammalian protein extraction reagent (M-PER) from the cell lines. The proteins were digested using the Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA) and complete protease inhibitor cocktails (Roche, Lewes, UK) according to the manufacturer’s protocols. The digested proteins were separated on 4–15% gradient sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The following primary antibodies were used: Cul4A (Abcam, Cambridge, MA, USA), ANXA10 (GeneTex), and β-actin (Sigma, St. Louis, MO, USA). After incubation with indicated secondary antibodies, the membranes were washed thoroughly and an enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for antigen-antibody detection. The relative intensities of protein bands were analyzed by densitometry using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA lysis buffer (50-mM Tris–HCl, 150-mM NaCl, 5-mM EDTA, 0.5% Nonidet P-40, and a protease and phosphatase inhibitor cocktail; Calbiochem, Darmstadt, Germany). Proteins were separated by SDS-PAGE and transferred to 0.45-μM polyvinylidene difluoride membranes (Millipore). The immunoblots were processed according to standard procedures using primary antibodies directed to EGFP, p21, GAPDH, HA, Flag, V5, HDAC3 (CST, Danvers, MA, USA), DsRed, Hsp70, p53, c-Myc, Cdc25A (Santa Cruz, Dallas, TX, USA), DDB1, Cul4A, Cul4B (Abcam, Cambridge, MA, USA), DCAF8 (Bethyl, Montgomery, TX, USA) and β-tubulin (Bioworld, Louis Park, China).
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein in HK-2 cells. The concentration was examined by the BCA method. After incubation with the loading buffer, the same amount of protein (30 mg) from each group was added into SDS-PAGE. The voltage was set to 120 volts. When the protein is sufficiently separated, it is transferred to the PVDF membrane. The current was set to 300 mA. After the membranes were blocked by QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China), primary antibodies (Cul4a, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; IκKα, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; IκBα, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; GAPDH, Abcam, Cambridge, MA, USA, Rabbit, 1:1000) were added and incubated at 4°C. The next day, the secondary antibody was used to incubate the membranes. The electrochemiluminescence (ECL) developer was added dropwise to develop imaging, and the grey value was semi-quantitatively analyzed according to Image J software.
7
Protein Expression and Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole protein was extracted by M‐PER Mammalian Protein Extraction Reagent added with Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA) and Complete Protease Inhibitor Cocktails (Roche, Lewes, UK). Proteins were separated on 7.5% SDS‐PAGE and transferred to Immobilon‐P membranes (Millipore, Billerica, MA, USA). The following primary antibodies were used: Cul4A (Abcam, Cambridge, MA, USA), cleaved PARP antibody (Cell Signaling, Danvers, MA, USA), p21 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TGF‐β inducible early gene‐1 (TIEG1; Abcam), transforming growth factor, beta‐induced (TGFBI; Abcam) and β‐actin (Sigma‐Aldrich). After antigen–antibody complexes were bound to secondary antibodies, an enhanced chemiluminescence blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used to detect antigen–antibody complexes.
8
Proteomic Profiling of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from tissue samples and cells using RIPA lysis buffer. The protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology, China). The standard western blot was performed in order to transfer protein onto PVDF membranes. Membranes were then incubated with an antibody against CUL4A (1:200, Abcam, UK), E-cadherin (1:100, Abcam, UK), N-cadherin (1:100, Abcam, UK), Vimentin (1:100, Abcam, UK), ZEB1 (1:500, Abcam, UK) and GAPDH (1:1000, Cell Signaling Technology, USA) overnight at 4°C. After washing with TBST, the membranes were incubated with a secondary antibody against mouse immunoglobulin G. The proteins were detected using ECL (Pierce Biotechnology, USA) according to the manufacturer's instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!