Fibroblasts were lysed in RIPA buffer containing 1 × complete protease inhibitor (Roche). Western blot analysis was performed with antibodies against Miro1 (WH0055288M1; Sigma–Aldrich, Munich, Germany), LC3-I/II (2775; Cell Signaling), Hsp60 (4870; Cell Signaling), Tom20 (sc-17764; Santa Cruz Biotechnologies), Rab9 (sc-74482; Santa Cruz Biotechnologies), MnSOD (ab13533; Abcam), anti-V5 (R960-25; Novex, R96125; Sigma–Aldrich), and β-actin (MA1-744; Thermo Scientific). Mitochondrial fractionation was performed as described previously (12 (link)).
Ma1 744
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The MA1-744 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a component that serves a core function in various scientific and research applications. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Tom20, by Santa Cruz Biotechnology (1 mentions)
Miro1, by Merck Group (1 mentions)
Ab13533, by Abcam (1 mentions)
Wh0055288m1, by Merck Group (1 mentions)
Hsp60, by Cell Signaling Technology (1 mentions)
Lc3 1 2, by Cell Signaling Technology (1 mentions)
Pa5 72344, by Thermo Fisher Scientific (1 mentions)
Pa5 13174, by Thermo Fisher Scientific (1 mentions)
Anti rabbit a3687, by Merck Group (1 mentions)
Ab5408, by Abcam (1 mentions)
Ab24758, by Abcam (1 mentions)
Ab817, by Abcam (1 mentions)
4336 bpc 100, by Bio-Techne (1 mentions)
Ecl chemiluminescence kit, by GE Healthcare (1 mentions)
Sc 2005, by Santa Cruz Biotechnology (1 mentions)
α sma ab32575, by Abcam (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Quantstudio 3 real time system, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
9 protocols using ma1 744
1
Mitochondrial Protein Analysis by Western Blot
2
Quantifying Liver Fibrosis Markers
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Liver fibrosis was evaluated using two different markers: hydroxyproline and α-SMA. The first one was determined according to previously described method [60 (link)]. The procedure utilized a volume of 0.5 mL of 20% liver homogenate which was kept in 1 mL of 6 M HCl for 8 h at 120 °C. A portion of the digested homogenate (25 μL) is added to 25 μL citrate-acetate buffer then 500 μL of chloramine T solution was added and finally the mixture is kept for 20 min at room temperature. Then, 500 μL Ehrlich’s solution was added and the mixture is incubated at 65 °C for 15 min. After cooling for 10 min, the color developed was spectrophotometrically measured at 550 nm. The results were presented as μg/g of wet tissue. In addition, α-SMA expression in formalin-fixed paraffin-embedded rat liver was assessed immunohistochemically using monoclonal antibody (MA1-744, ThermoFisher, Loughborough, UK).
3
Protein Interaction and Regulation Analysis
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Western blot analysis: The following primary antibodies were used: PARP1 C terminal, rabbit polyclonal (#39561, Active Motif, Carlsbad, CA, USA); Actin, mouse monoclonal (MA1-744, Thermo Fisher Scientific, Waltham, MA); H5 (Ser2P), mouse monoclonal (ab24758, Abcam, Cambridge, MA, USA); IntS3, rabbit polyclonal (PA5-72344, Thermo Fisher Scientific, Waltham, MA, USA); AF9, rabbit polyclonal (PA5-13174, Thermo Fisher Scientific, Waltham, MA, USA), Cyclin T1, rabbit polyclonal (PA5-24163, Thermo Fisher Scientific, Waltham, MA, USA); PAR rabbit polyclonal (4336-BPC-100, Trevigen, Gaithersburg, MD, USA). Secondary antibodies used: anti-rabbit (A3687) and anti-mouse (A3562) IgG (whole molecule) alkaline phosphatase antibody (Sigma).
Co-immunoprecipitation and chromatin immunoprecipitation (ChIP): PARP1, rabbit polyclonal (#39561, Active Motif, Carlsbad, CA, USA); H5 (Ser2P), mouse monoclonal (ab24758, Abcam, Cambridge, MA, USA); 4H8, mouse monoclonal (ab5408, Abcam, Cambridge, MA, USA); 8WG16, mouse monoclonal (ab817, Abcam, Cambridge, MA, USA); IntS3, rabbit polyclonal (PA5-72344, Thermo Fisher Scientific, Waltham, MA, USA); AF9, rabbit polyclonal (PA5-13174, Thermo Fisher Scientific, Waltham, MA, USA).
Co-immunoprecipitation and chromatin immunoprecipitation (ChIP): PARP1, rabbit polyclonal (#39561, Active Motif, Carlsbad, CA, USA); H5 (Ser2P), mouse monoclonal (ab24758, Abcam, Cambridge, MA, USA); 4H8, mouse monoclonal (ab5408, Abcam, Cambridge, MA, USA); 8WG16, mouse monoclonal (ab817, Abcam, Cambridge, MA, USA); IntS3, rabbit polyclonal (PA5-72344, Thermo Fisher Scientific, Waltham, MA, USA); AF9, rabbit polyclonal (PA5-13174, Thermo Fisher Scientific, Waltham, MA, USA).
4
Western blot analysis of liver proteins
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Whole liver tissue was lysed with ice cold lysis buffer for SDS–PAGE (1 M NaCl, 0.01 M EGTA, 0.5 M EDTA, 1 M NaH2PO4, 1 M Tris, 1 M NaF, 10× Triton, 0.1 M PMSF in EtOH, 1 mM Na3VO4). The protein lysat was denaturated at 95°C for 5 min in double-strength sodium dodecyl sulfate sample buffer with dithiothreitol before resolving in 10%% SDS–PAGE. Primary antibody incubation was performed with α-SMA (ab 32575, Abcam, Cambridge, United Kingdom) and β-Actin (MA1-744, Thermofisher, Waltham, Boston, MA, United States) antibodies. For detection secondary HRP-linked antibodies against rabbit IgG (7074S, Cell signaling, Frankfurt, Germany) or mouse IgG (sc-2005, Santa Cruz, Heidelberg, Germany) were used. To visualize the antigen-antibody complex the ECL Chemiluminescence Kit (GE Healthcare, Buckinghamshire, United Kingdom) was used.
5
Epigenetic Regulation Assay Protocol
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For western analysis, cells were lysed in radioimmune precipitation buffer and separated by SDS/PAGE. Antibodies to actin (MA1‐744; Thermo Fisher; Waltham, MA, USA), ubiquitin H2A‐K119 (3240; Cell Signaling Technology; Danvers, MA, USA), H3K27me3 (9733; Cell Signaling Technology), BMI1 (6964; Cell Signaling Technology), EZH2 (5246; Cell Signaling Technology), DNMT3B (ab2851; Abcam; Cambridge, UK), and DNMT1 (SC‐20701; Santa Cruz Biotechnology; Dallas, TX, USA) were used. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen), and complementary DNAs were synthesized using iScript Reverse Transcription Supermix (Bio‐Rad Laboratories; Hercules, CA, USA). Quantitative real‐time PCR assays were performed with iTaq Universal SYBR Green Supermix (Bio‐Rad Laboratories) and the QuantStudio 3 Real‐time System (Thermo Fisher). In all cases, gene expression was normalized to β‐actin. Primers for RT‐PCR are provided in Table S1 .
6
Immunoblotting of Ndufs1 and Ndufb10
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Immunoblotting was performed as previously described [53 (link)]. Mouse ESCs or STO cells were trypsinized, collected, and resuspended with Laemmli loading buffer. After 12% or 8% SDS-PAGE electrophoresis proteins were transferred to the nitrocellulose membrane with semi-dry transfer (Helikon, Moscow, Russia), the Ndufs1 and Ndufb10 proteins were detected with rabbit antibodies (PA5-22309, Thermo Fisher Scientific, Waltham, MA, USA) (1:1000 dilution) and (ab196019, Abcam, Cambridge, UK) (1:1000 dilution), respectively. For a loading control, mouse anti-beta-actin antibodies (MA1-744, Thermo Fisher Scientific, Waltham, MA, USA) (1:5000 dilution) were used. Anti-mouse and rabbit HRP-conjugated antibodies were used as secondary antibodies (#115-036-062 and #111-036-045, Jackson ImmunoResearch, West Grove, PA, USA) (1:5000 dilution). Chemiluminescence was detected with ChemiDoc Touch (Bio-Rad Laboratories, Hercules, CA, USA).
7
Immunoblot Analysis of Mouse iPSCs
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Immunoblotting was performed as previously described [73 (link)]. Mouse iPSCs pellets were resuspended with Laemmli loading buffer. After 10% SDS-PAGE electrophoresis, and the protein transfer to the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) with semi-dry transfer (Helikon, Russia), the TPH and beta-actin (a loading control) proteins were detected with mouse monoclonal antibodies T0678 (Sigma-Aldrich, St. Louis, MO, USA) and MA1-744 (Thermo Fisher Scientific), respectively. Anti-mouse IgG HRP-conjugated antibodies were used as secondary antibodies (#115-036-062 and #111-036-045, Jackson ImmunoResearch, USA). Chemiluminescence was detected with a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).
8
Immunoblotting for Rad53 and Rad52
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Briefly, 2.0 × 107 cells were pelleted and protein was isolated and immunoblotting was performed6 . Rad53 and Rad52 were detected using anti-Rad53 (Abcam-ab104232) and anti-Rad52 (gift from B. Pfander) antibodies, both diluted 1/10,000. Actin was detected using anti-Actin antibody (ThermoFisher-MA1-744) diluted 1/5000. Chemiluminescence was captured using autoradiography and VersaDoc imager to ensure ideal exposure in linear range.
9
Genetic Manipulation and RNA-DNA Detection
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Endogenous genes were deleted as described (23 (link)). Yeast strains and primers used are listed (Supplementary Tables S3 and S4). R-loop detection via direct pull downs employed the anti-RNA–DNA antibody from Kerafast (cat# ENH001), which exhibited lower enrichments but higher specificity compared to antibodies that we used previously in chromatin immunoprecipitation (ChIP) (12 (link)). Anti-RNAPII, anti-diAcH3K9/14, anti-TAP and anti-β-Actin antibodies were purchased from BioLegend (665004), Millipore (06-599), Sigma (P1291-500UL) and Thermo Scientific (MA1-744), respectively. Anti-GFP was a kind gift from D. Moazed (Harvard University). The Stm1 YEplac181 plasmid or empty vector control were kind gifts from F.B. Johnson (University of Pennsylvania) (24 (link)).
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