For CHX-chase experiments, UBC12-knockdown cells and control cells were treated with 50 μg/mL CHX (sigma) for indicated time points. Cell lysates were prepared for immunoblotting analysis using antibodies against UBC12, UBA3, Cullin1, Cullin2, Cullin5, p21 (abcam), NAE1, Cullin3, Cullin4a, p27, Wee1, p-H3, NEDD8 (Cell Signaling, Boston, MA), Cullin4b (protein Tech). β-actin (protein Tech) was used as the loading control.
Cullin4b
Manufactured by Proteintech
Cullin4B is a core component of the Cullin-RING E3 ubiquitin ligase complex. It functions as a scaffold protein, facilitating the ubiquitination of substrate proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (2 mentions)
β actin, by Proteintech (2 mentions)
Cullin2, by Abcam (2 mentions)
Cullin5, by Abcam (2 mentions)
Cullin1, by Abcam (2 mentions)
Cullin4a, by Cell Signaling Technology (2 mentions)
Cullin3, by Cell Signaling Technology (2 mentions)
Nedd8, by Cell Signaling Technology (1 mentions)
Cullin3, by Abcam (1 mentions)
P h2ax, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Nedd8, by Abcam (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Aurka, by Abcam (1 mentions)
Ubiquitin, by Cell Signaling Technology (1 mentions)
Xbp1s, by Cell Signaling Technology (1 mentions)
β actin hrp, by Abcam (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
5 protocols using cullin4b
1
CHX-chase analysis of UBC12 depletion
2
Western Blot Analysis of Protein Expression
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For Western blot, cell lysates (30 mg) were loaded on SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore), which were then incubated with the indicated primary antibodies overnight. Corresponding secondary antibodies were incubated with the membranes for 1 h, and the membranes were then photographed with a Tanon 5200 visualizer (Shanghai, China). Primary antibodies to the following proteins were used: UBC12, UBA3, cullin1, cullin2, cullin5, p21, NOXA (Abcam), NAE, cullin3, NEDD8, cullin4A, p27, Wee1, p-H3, ORC1, CDT1, p-H2AX, t-H2AX, ATF4, CHOP, DR5, cleaved-PARP, cleaved-caspase 8, and cleaved-caspase 3 (Cell Signaling, Boston, MA); cullin4B (Protein Tech); and β-actin (Protein Tech). For CHX-chase assays, cells were treated with 50 μg/mL CHX (Sigma) for the indicated times, and the band density in Western blot was quantified in Image J software.
3
Protein Extraction and Western Blot Procedure
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Proteins from fresh tissues and cell lines were treated with RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined by the BCA method, and proteins were separated by 8%–12% SDS–PAGE and transferred to polyvinyl difluoride membranes. The membranes were incubated overnight at 4 °C in the corresponding primary antibodies including AURKA (Abcam, ab1287), GAPDH (CST, #5174), SIN1 (CST, #12860), AKT (CST, #4691), P-AKT (CST, #4060), MYC-TAG (CST, #2276), HA-TAG (CST, #2376), Ubiquitin (CST, #3936), Cullin4B (ProteinTech, 12916), HIS-TAG (CST, #12698), DYKDDDDK TAG (CST, #14793). Subsequently, luminescence detection was performed using ECL reagents after incubation with the corresponding secondary antibodies.
4
Quantitative Western Blotting Technique
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Western blot was performed using 10–30 μg of total protein lysates or 20 μg of nuclear extracts. The following antibodies were used in this study: XBP1s (Cell Signaling, 83418), CHOP (Cell Signaling, 2895), PARP (Cell Signaling, 9532), ubiquitin (Cell Signaling, 3933), Cullin1 (Cell Signaling, 4995), Cullin3 (Cell Signaling, 2759), Cullin4B (Proteintech, 12916-1-AP), SPOP (Proteintech, 16750-1-AP), Flag (Sigma, F1804 and F7425), Myc (Cell Signaling, 2278 and 2276), HA (Sigma, H3663; and Cell Signaling, 3724), βActin-HRP (Abcam, ab49900), and HSP90 (Santa Cruz Biotechnology, sc7947). Secondary antibodies—donkey antirabbit or mouse IgG HRP (Jackson ImmunoResearch)—were used at 1:6000 dilution. Western blot membranes were developed using the Clarity Western ECL substrate (Bio-Rad), and signal was detected with an ImageQuant LAS4000 luminescent imager (General Electric). Quantification was performed using ImageQuantTL software (General Electric).
5
Cullin-RING Ubiquitin Ligase Toolkit
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Antibodies specific to C-PARP (Cell Signaling Technology, 5625), Cullin1 (Abcam, 75817), Cullin2 (Abcam, 166917), Cullin3 (Cell Signaling Technology, 2759), Cullin4A (Cell Signaling Technology, 2699), Cullin4B (Proteintech, 12916-1-AP), Cullin5 (Abcam, 184177), NAE (Cell Signaling Technology, 14321), NOXA (Cell Signaling Technology, 14766), P27 (Cell Signaling Technology, 3686), RBX1 (Abcam, 133565), UBA3 (Abcam, 124726), UBE2F (Abcam, 12932), and UBE2M (Abcam, 109507) were purchased commercially.
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