Paraffin sections were collected and slides prepared as described above. For SOX9, OSX, OPN, KI67, and RUNX2 antibodies, antigen retrieval was performed through boiling the slides in citrate buffer (10 mM trisodium citrate, pH 6.0; 0.05% Tween20) for 20 minutes. For COL1, COL2a, COLX, MMP13, VEGFA antibodies, samples were incubated in hyaluronidase for 30 minutes at 37°C. Next, slides were blocked in 5% donkey serum in PBS with 0.05% Triton X-100 (PBSX) for one hour. Slides were then incubated with the primary antibody overnight at 4°C. The following antibodies were used for immunofluorescence microscopy: COL IIa (Abcam,ab185430, 1:100); COLX (Abcam, ab5832, 1:50); COL I (Abcam,ab88147; GR3225500–1, 1:100); MMP13 (Abcam, ab39012, 1:100); RUNX2 (Abcam,ab76956, 1:200), IB4 (Thermo Fisher Scientific cat: VECTB1205, 1:500), SOX9 (MilliporeSigma, AB55535, 1:200); OPN (SCBT, sc22536-R, 1:100); OSX (SCBT, sc21742, 1:100); KI67 (Bethyl, IHC00075, 1:100).
Ab5832
Manufactured by Abcam
Sourced in United Kingdom
Ab5832 is a laboratory equipment product manufactured by Abcam. It is designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ab56574, by Abcam (2 mentions)
Ab39012, by Abcam (1 mentions)
Ab88147, by Abcam (1 mentions)
Ab185430, by Abcam (1 mentions)
Phospho stat3 ser727, by Cell Signaling Technology (1 mentions)
Total stat3, by Cell Signaling Technology (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Ab54708, by Abcam (1 mentions)
Ab54593, by Abcam (1 mentions)
Ab11825, by Abcam (1 mentions)
Al125c, by PerkinElmer (1 mentions)
Ez link sulfo nhs biotin kit, by Thermo Fisher Scientific (1 mentions)
Envision microplate reader, by PerkinElmer (1 mentions)
Ez magna rip kit, by Merck Group (1 mentions)
N cadherin, by Proteintech (1 mentions)
Ab127169, by Abcam (1 mentions)
Anti β actin, by Boster Bio (1 mentions)
Anti pcna, by Proteintech (1 mentions)
Goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
12 protocols using ab5832
1
Immunofluorescence Analysis of Chondrocyte Markers
2
Western Blot Analysis of STAT3 Signaling
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Whole cell lysates were prepared by lysis in buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol, 1% Triton X‐100, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM EGTA, 1.5 mM MgCl2, 0.1 mM sodium orthovanadate, and complete EDTA‐free protease inhibitors (Roche Applied Science). Primary antibodies used were phospho‐Tyr705‐STAT3 (1:2,000; #9145, Cell Signaling), total STAT3 (1:1,500; #9132 or 9139, Cell Signaling), phospho‐Ser727‐STAT3 (1:1,000; #9134, Cell Signaling), STAT3α (1:1,000; C‐20, Santa Cruz), α‐tubulin (1:2,000; clone 6G7 deposited by Halfter WM to Developmental Studies Hybridoma Bank), iNOS (1:3,000; Clone 6, BD Transduction Laboratories), hnRNP A1 (1:1,000; ab5832, Abcam), p65 (1:1,000; 06‐418, EMD Millipore), and 3A2 [anti‐HuR, 1:10,000 (Gallouzi et al, 2000 )].
3
Quantification of Arginine Methylation
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TCR-activated PBMCs employed for the MethylScan study were used to generate lysates as described above for Western blot analysis. Protein concentrations were determined using the BCA Protein Assay Kit. The anti-ADMA antibody (CST, clone D10F7A10) was biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific, #21326) according to the manufacturer’s instructions. 1 µg of lysates, in triplicates, was incubated with the biotinylated anti-ADMA antibody, at a concentration of 0.5 µg/mL, and antibodies specific to ALYREF (Abcam, #ab6141), DHX9 (Abcam, #ab54593), EIF4H (Abcam, #ab77455), EWSR1 (Abcam, #ab54708), G3BP1 (Abcam, #ab56574), HNRNPA1 (Abcam, #ab5832), KHDRSB1 (Abcam, #ab56836), SFPQ (Abcam, #ab11825) and TPR (Abcam, #ab58344) at a concentration of 1 µg/mL. After a 45-min incubation at room temperature, streptavidin-coated acceptor beads (Perkin Elmer, #AL125C) and anti-mouse IgG-coated donor beads (Perkin Elmer, #AS104) were added to respective wells of 96 well microplates (Corning, #3693), at a final concentration of 20 µg/mL and incubated for an additional 45 min at room temperature. All solutions were prepared in AlphaLISA universal buffer (Perkin Elmer, #AL001F) and microplates were shaken after each addition and centrifuged prior to luminescence detection on an Envision microplate reader (Perkin Elmer).
4
EV-mediated hnRNPA1-PROX1 mRNA Interaction
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To identify the interaction between EV-packaged hnRNPA1 and PROX1 mRNA, RIP assays were performed using an EZ-Magna RIP kit (Millipore, 17-701) according to the manufacturer’s instructions. Briefly, 2 × 107 HLECs treated with 10 μg/mL EVs were harvested and lysed in cell lysis buffer containing RNase and protease inhibitors. Then, magnetic bead–coupled anti-hnRNPA1 antibodies (Abcam, ab5832) or normal rabbit IgG as the negative control were added to the cell lysate and immunoprecipitated at 4°C overnight. Next, the magnetic beads were washed with RIP washing buffer. The combined RNA was extracted for qRT-PCR analysis, in which U1 was used as the nonspecific control. Supplemental Tables 6 and 7 list the primer sequences and the antibodies used, respectively.
5
Comprehensive Protein Expression Analysis
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Following antibodies were used in western blot: anti-CCDC50 (ab127169, Abcam), anti-ZNF395 (11759–1-AP, Proteintech), anti-HnRNP A1 (ab5832, Abcam), anti-β-actin (BM0627, Boster), anti-PCNA (10205–2-AP, Proteintech), anti-Cyclin D1 (60186–1-Ig, Proteintech), N-Cadherin (22018–1-AP, Proteintech), anti-Vimentin (BM0135, Boster), anti-ZEB1 (3396, Cell Signaling Technology), anti-VEGF (19003–1-AP, Proteintech), goat anti-mouse IgG HRP-linked whole antibody (31,430, Thermo Scientific), and goat anti-rabbit secondary antibody (31,460, Thermo Scientific).
6
Immunoprecipitation and RNA-Protein Complexes
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Immunoprecipitation was performed by incubating 1 µg hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) or Fyn mouse monoclonal antibody (P2992; Sigma-Aldrich) with 500 µg extracted proteins overnight at 4°C with constant rotation. The immunocomplexes were captured by adding protein A agarose (15918014; Invitrogen/Thermo Fisher Scientific) for 1 h at 4°C with constant rotation. The immunoprecipitates were analyzed by western blotting.
For RNA-protein immunoprecipitation, 1 mg extracted proteins were pre-cleared with 20 µl protein A/G plus agarose (20423; Invitrogen/Thermo Fisher Scientific) and then incubated with 5 µg of hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology), SF2/ASF rabbit polyclonal antibody (ab38017; Abcam) or hnRNP A1 mouse monoclonal antibody (ab5832; Abcam) overnight at 4°C with constant rotation. The co-precipitated RNA was then extracted by phenol (AM9712; Thermo Fisher Scientific)/chloroform (1.02444; Sigma-Aldrich) and used for further analysis.
For RNA-protein immunoprecipitation, 1 mg extracted proteins were pre-cleared with 20 µl protein A/G plus agarose (20423; Invitrogen/Thermo Fisher Scientific) and then incubated with 5 µg of hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology), SF2/ASF rabbit polyclonal antibody (ab38017; Abcam) or hnRNP A1 mouse monoclonal antibody (ab5832; Abcam) overnight at 4°C with constant rotation. The co-precipitated RNA was then extracted by phenol (AM9712; Thermo Fisher Scientific)/chloroform (1.02444; Sigma-Aldrich) and used for further analysis.
7
Microscopic Analysis of SMPX-V5 Overexpression
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For microscopic analyses, HeLa cells were plated on coverslips in 24-well plates and transfected with SMPX-V5 constructs (500 ng/well) using FuGENE 6. Three days after transfection, the cells were treated with 20 µM MG132 (Sigma-Aldrich) for 2 h or left untreated. Cells were washed with PBS and fixed with 4% PFA for 15 min. Immunofluorescence stainings were performed with the following antibodies: V5 mouse mAb (RRID:AB_2556564), V5 rabbit pAb (Sigma-Aldrich AB3792, RRID:AB_91591), TIA1 goat pAb (Abcam ab61700, RRID:AB_945832), TIAL1 rabbit pAb (Abcam ab26257, RRID:AB_470826), TIAL1 rabbit mAb D32D3 (RRID:AB_10839263), G3BP mouse mAb (Abcam ab56574, RRID:AB_941699), HNRNPA1 mouse mAb 9H10 (Abcam ab5832, RRID:AB_305145), eIF3η goat pAb (RRID:AB_671941), and Oligomer A11 rabbit pAb (Thermo Fisher AHB0052, RRID:AB_2536236). Nascent proteins were labeled and detected with the Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. Images were acquired with Zeiss Axio Imager M2 using 40× NA 1.30 and 20× NA 0.80 objectives, or with a Leica TCS SP8 confocal microscope using a 40× NA 1.10 objective (Leica Microsystems).
8
Western Blot Analysis of Muscle Protein
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For western blotting analysis, the muscle biopsy from IV to 11 was homogenized in the Laemmli sample buffer and heated at 95°C for 5 minutes. The soluble fraction after centrifugation (13,000 × g, 10 minutes) was collected and used for SDS-PAGE using 4%–20% TGX gradient gels and Bio-Rad Mini-PROTEAN system (Bio-Rad Laboratories, CA). After separation, the proteins were transferred onto PVDF membranes using the Bio-Rad TransBlot Turbo device. The membranes were incubated with anti-HNRNPA1 (Abcam ab5832) antibody at 1:10,000, diluted in 5% nonfat milk powder/TBST, overnight at +8°C. After HRP-conjugated secondary antibody incubation, ECL reaction (SuperSignal West Femto, Thermo Fisher Scientific) and Bio-Rad ChemiDoc reader were used for detection of bands. The gels were recovered after transfer and stained with Coomassie, and the myosin heavy chain bands were used as loading control.
9
ADMA Quantification in Cancer Cell Lysates
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Lysates from Toledo and Jurkat cancer cells and PBMCs treated with GSK3368712 were generated as described above for Western blot analysis and protein concentrations were assessed using the BCA Protein Assay Kit. 1 µg of lysates, in triplicates, was incubated with a custom anti-ADMA antibody (clone D10F7A10; provided to GSK by CST as a beta product) and an antibody specific to hnRNP-A1 (Abcam, #ab5832), at a final concentration of 1 µg/mL in AlphaLISA universal buffer for 1 h at room temperature. Anti-rabbit-IgG-coated acceptor beads (Perkin Elmer, #AL104) were added at a final concentration of 20 µg/mL and incubated for 1 h at room temperature. Anti-mouse IgG donor-coated beads (Perkin Elmer, #AS104) were added at a final concentration of 40 µg/mL and incubated for an additional 2 h at room temperature. Microplates were shaken after each addition and centrifuged prior to luminescence detection on an Envision microplate reader.
10
Polyubiquitin and p97 Protein Analysis
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The following antibodies were used in this study (WB, Western blot (Bio-Rad [Hercules, CA, USA]). IF, immunofluorescence): K48 linkage-specific polyubiquitin (WB, 1:4000. IF, 1:500. MilliporeSigma [Burlington, MA, USA], Apu2), HA-peptide (WB, 1:2000. IF, 1:500. Roche, 3F10), p97 (WB, 1:7000. Abcam [Cambridge, England], ab109240), α-tubulin (WB, 1:5000. MilliporeSigma [Burlington, MA, USA], T5168), α-GAPDH (WB, 1:10,000. Proteintech [Rosemont, IL, USA], 60004-1-Ig), α-Ufd1 (WB, 1:1000. Cell Signaling [Danvers, MA, USA], 13789), α-Npl4 (WB, 1:1000. Cell Signaling, 13489), α-UBXD1 (WB, 1:500. Bethyl Laboratories [Montgomery, TX, USA], A302-931A), α-hnRNPA1 (WB, 1:2000. Abcam [Cambridge, England], ab5832), α-emerin (WB, 1:4000. Cell Signaling [Danvers, MA, USA], 30853S), α-calnexin (WB, 1:2000. Abcam [Cambridge, England], 75801), FLAG peptide (WB, 1:4000. IF, 1:500. MilliporeSigma [Burlington, MA, USA], F3165), rabbit IgG HRP conjugate (WB, 1:10,000. SouthernBiotech [Birmingham, AL, USA], 4030-05), mouse IgG HRP conjugate (WB, 1:20,000. SouthernBiotech [Birmingham, AL, USA], 1030-05), rabbit and mouse IgG Alexa488 conjugates (IF, 1:700. Invitrogen [Waltham, MA, USA], A11008 and A28175), and rabbit and mouse IgG Alexa568 (IF, 1:700. Invitrogen [Waltham, MA, USA], A-11011 and A-11004).
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