Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.30 (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.
Ab24733
Manufactured by Abcam
Sourced in United States
Ab24733 is a type of lab equipment. It is designed for use in scientific research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Ab14748, by Abcam (2 mentions)
Ab2907, by Abcam (2 mentions)
Ab14711, by Abcam (1 mentions)
Ab14745, by Abcam (1 mentions)
Ab14705, by Abcam (1 mentions)
Ab14744, by Abcam (1 mentions)
Ab110242, by Abcam (1 mentions)
Ab14714, by Abcam (1 mentions)
Ab110258, by Abcam (1 mentions)
Ab7291, by Abcam (1 mentions)
A1978, by Merck Group (1 mentions)
P0260, by Agilent Technologies (1 mentions)
P0399, by Agilent Technologies (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Ab6789, by Abcam (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Lenalidomide, by MedChemExpress (1 mentions)
Bortezomib, by MedChemExpress (1 mentions)
Cyclophosphamide, by MedChemExpress (1 mentions)
Ab205719, by Abcam (1 mentions)
8 protocols using ab24733
1
Western Blot Analysis of Skeletal Muscle
2
HABP1 Protein Expression Analysis in Tissue Samples
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Frozen tissue specimens were homogenized in radioimmunoprecipitation assay (RIPA) buffer consisting of 1% protease inhibitor mixture. The mixture was centrifuged at 12,000× g for 15 minutes at 4°C, and the supernatant was obtained. Total proteins were quantified, and 30 μg of protein per sample was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride film (Bio-Rad, Carpinteria, CA, USA). The membranes were blocked by 2% bovine serum albumin at 37°C for 1 hour and incubated with primary antibodies, anti-HABP1 (1:800 dilution, ab24733; Abcam, Cambridge, MA, USA), overnight at 4°C. After a standard washing, the film was incubated with horseradish peroxidase-labeled secondary antibody for 1 hour at room temperature and washed again. The blots were stained using a SuperSignal Kit (Pierce, Rockford, IL, USA) and imaged by a charge-coupled camera LAS4000 (Fujifilm, Tokyo, Japan). The experiment was repeated in triplicate.
3
Quantifying C1QBP Protein Expression
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The RIP assay for C1QBP was performed at room temperature to quantify the protein expression using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–701, Millipore, Billerica MA, USA) following the manufacturer’s instructions. TU212 cells were incubated using magnetic beads conjugated with C1QBP-specific antibody (ab24733, Abcam, Cambridge, MA, USA) or control IgG (ab6789, Abcam, Cambridge, MA, USA). The immunoprecipitated RNA was reverse-transcribed into the cDNA and was amplified using qPCR to identify any circMTCL1. The primers are listed in Table S10 .
4
Evaluating C1q Receptor Expression
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WB was performed as previously described (17 (link)). Each experiment was repeated at least three times, with a representative experiment shown. The antibodies were as follows: anti-gC1qR antibody (Abcam, ab24733), anti-cC1qR antibody (Abcam, ab2907), anti-IGF2BP3 antibody (Abcam, ab177477), anti-β-actin antibody (Cell Signaling Technology, 4967S), and secondary horseradish-peroxidase-conjugated antibodies (Abcam, ab205719, ab205718). Five clinical drugs (bortezomib, ixazomib, lenalidomide, pomalidomide, and cyclophosphamide) were purchased from Med Chem Express, USA.
5
Immunophenotyping of C1q Receptors
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To analyze the membrane gC1qR and cC1qR, cells were incubated in PBS with primary antibodies (anti-gC1qR and anti-cC1qR, Abcam, ab24733, and ab2907) after sorting at 4°C for 20 min. Then, the diluted secondary antibodies, which were anti-rabbit IgG [Alexa Fluor 647 conjugated, allophycocyanin (APC) fluorochromes, red fluorescence channel] (Cell Signaling Technology, Danvers, MA, USA, 4410S) and anti-mouse IgG [fluorescein isothiocyanate (FITC) conjugated, FITC fluorochromes, and green fluorescence channel) (Jackson Immuno Research, West Grove, PA, USA, 115-095-003), were used to stain the cells at 4°C for 20 min. Finally, the expression of the surface C1qRs was measured by flow cytometry (FC) (BD FACSCanto II, Franklin Lake, New Jersey, USA). The mean fluorescence intensity (MFI) of each fluorescence was measured using FlowJo V10 (BD FACSCanto II, USA). Each cell-based experiment was repeated at least three times.
6
C1QBP and β-catenin Interaction Assay
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Co-IP assays for C1QBP and β-catenin were performed at room temperature. Lysates containing ten million cells were incubated with 1-μganti-C1QBP antibody (ab24733, Abcam, Abcam, Cambridge, MA, USA) and anti-β-catenin antibody (ab32572, Abcam, Abcam, Cambridge, MA, USA) at 4 °C overnight with slow rotation. The antigen-antibody complexes were retrieved using protein A beads (8687S, CST, Danvers, MA, USA) and then washed three times with pre-cooled phosphate-buffered saline. IP complexes were eluted with Laemmli buffer and subjected to WB.
7
RIP Assay for lncMtDloop Enrichment
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RIP was performed using a Magnetic RIP RNA-Binding Protein Immunoprecipitation (Millipore, 17-700) in accordance with the manufacturer's guidelines. The primary cultured neurons were treated with ice-cold PBS and IP lysis buffer (Thermo Fisher Scienti c, 87787) supplemented with RNase inhibitors and protease inhibitor cocktail. The supernatants were incubated with the anti-p32 (ab24733, Abcam) antibody or control mouse IgG with rotation at 4°C overnight, and then protein A/G beads were added to each group. After the incubation at 4°C overnight, we collected the precipitates and extracted RNA by proteinase K-chloroform method. The enrichment of lncMtDloop was detected using qRT-PCR.
8
Comprehensive Western Blot Analysis of Brain Samples
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Samples from mouse brain and primary cell culture were collected and homogenized in RIPA buffer containing protease inhibitors and phosphatase inhibitors. After centrifugation, supernatants were heated to 95℃ for 10 min, and resolved on 6-12% polyacrylamide precast gels. The gel was transferred to PVDF membrane at 250 mA for 90 min at 4℃ and blocked for 1 hr at room temperature in 5% BSA in Tris buffered saline with 0.05% Tween 20. Membranes were incubated in primary antibody overnight at 4℃. Antibodies used for western blot were: anti-6E10 (803001, BioLegend), anti-p32 (ab24733, Abcam), anti-ATP5a (ab14748, Abcam), anti-LC3B (ab51520, Abcam), anti-PINK1 (ab23707, Abcam), anti-parkin (#2132, Cell Signaling), anti-AT8 (MN1020, Thermo Fisher Scienti c), anti-AT180 (MN1040, Thermo Fisher Scienti c), anti-HT7 (MN1000, Thermo Fisher Scienti c), anti-TAU-5 (AHB0042, Thermo Fisher Scienti c), anti-β-actin (ab8227, Abcam), anti-COX IV (ab33985, Abcam), anti-Stxb1 (ab183722, Abcam), anti-Tppp (ab92305, Abcam), anti-Aly/Ref (ab202894, Abcam), anti-ATP5D (ab174438, Abcam), anti-TFAM (ab252432, Abcam). All blots were imaged using HRP-conjugated secondary antibodies. After three washes for 10 min each, the uorescence signals were quanti ed using Image J software.
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