SDS-PAGE and immunoblots were performed as previously described(20 (link)) using antibodies for β-catenin (C2206; Sigma, St. Louis, MO), tubulin (sc-5546; Santa Cruz Biotechnology, Santa Cruz, CA), VDR (sc-13133), E-cadherin (sc-21791), DKK-1 (GTX62902; GeneTex, Irvine, CA), LRP6 (CST3395; Cell Signaling Technology, Danvers, MA), RXRα (CST3085), RXRβ (CST8715), LDLRAP1 (C20125; LSBio, Seattle, WA) or LC3B (ab48394; Abcam, Cambridge, MA). For LRP6 and LC3B immunoblots, lysates were resolved on 4-20% gradient gels. Co-immunoprecipitations were performed as previously described(21 (link)). Briefly, AsPC-1 lysates were pre-cleared with A/G-PLUS agarose beads (Santa Cruz; sc-2003) and immunoprecipitated using antibody to β-catenin (BD Transduction Laboratories; 610153), VDR (Santa Cruz; sc-1008) or control isotype-matched IgG (Santa Cruz; sc-2025 or Abcam ab46540). After multiple washes, immune complexes were boiled in 6× SDS-load dye, resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting.
Sc 1008
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-1008 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab48394, by Abcam (1 mentions)
C2206, by Merck Group (1 mentions)
A g plus agarose beads, by Santa Cruz Biotechnology (1 mentions)
Sc 2025, by Abcam (1 mentions)
Sc 5546, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by GeneTex (1 mentions)
Minute total protein extraction kit, by Invent Biotechnologies (1 mentions)
Amersham ecl reagent, by Cytiva (1 mentions)
Anti succinyllysine antibody, by PTM Biolabs (1 mentions)
3 protocols using sc 1008
1
Protein Interaction Analysis by Co-Immunoprecipitation
2
Protein Expression Analysis of Metabolic Regulators
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Protein samples were isolated from tissue and cells using the Minute total protein extraction kit (Invent, SD001). Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Sigma Co., Ltd., Burlington, MA, United States). The membranes were probed with antibodies against SIRT3 (10099-1-AP, 1:500), LCAD (17526-1-AP, 1:300), Complex I (12444-1-AP, 1:1,000), Complex III (14865-1-AP, 1:1,000), Complex IV (18443-1-AP, 1:2,000), FAT/CD36 (18836-1-AP, 1:500), CPT-1β (22170-1-AP, 1:500), and GADPH (60004-1-Ig, 1:5,000) purchased from Proteintech as well as with an antibody against MTTP from Absin (abs136111a, 1:1,000) and antibodies against VDR (sc-1008, 1:1,000) and GLUT4 (sc-53566, 1:500) purchased from Santa Cruz. Following overnight primary antibody incubation, membranes were incubated with a secondary antibody (ZSGB-Bio, Beijing, China) for 1 h. The protein bands of interest were detected using Amersham ECL reagents (Cytiva, Little Chalfont, United Kingdom).
3
Identifying VDR-Succinyl Lysine Interactions
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VDR and succinylated proteins were precipitated from WAT by adding anti-VDR or ies in WT mice. The antibody of anti-VDR (sc-1008) was purchased from Santa Cruz Biotechnology (CA, USA). The anti-succinyl-lysine antibody was obtained from PTM Biolabs. After incubation in blocking buffer at 1:1000 for 4 h at 4 °C, 10 μl of protein A-Sepharose beads were added, and incubated with moderate stirring at 4 °C for 12 h. The beads were then washed, and the immunoprecipitated protein complex was loaded into 10% SDS-PAGE gel, electrophoresed and processed for western blotting. To identify a molecular interaction between VDR and succinyl-lysine, the VDR-immunoprecipitating complex was blotted for succinyl-lysine, and succinyl-lysine -immunoprecipitating complex blotted for VDR.
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