Fresh whole testes were homogenized in a non-denaturing lysis buffer (50 mM Tris-HCl, 170 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% NP-40) containing protease inhibitor cocktail cOmplete, Mini (Roche). The lysates were sonicated using Bioruptor (Diagenode, Liège, Belgium) at 2 × 30 s. After incubation on ice, the lysates were centrifuged at for 25 min 10 000 × g. One milligram of protein was precleared using 2 μg of normal rabbit IgG and 20 μl of Dynabeads Protein G (Invitrogen, Carlsbad, CA, USA). The lysate was then incubated with 2 μg of E2F3 C-18 antibody (sc-878, Santa Cruz) overnight at 4 °C. A control sample was prepared by adding 8 μg of E2F3 blocking peptide (sc-878P, Santa Cruz) to the reaction. Fifty microlitres of Dynabead Protein G was used to pull down the complexes. The immunoprecipitate together with input lysate was loaded to Mini-Protean TGX 4-20% SDS-PAGE gel (Bio-Rad). RB was detected from the upper part of the blot with mouse monoclonal anti-RB (MAB3186, Merck Millipore (Chemicon), Billerica, MA, USA), and E2F3 was detected from the lower part of the blot with E2F3 C-18 (sc-878, Santa Cruz).
Mini protean tgx 4 20 sds page gel
Manufactured by Bio-Rad
The Mini-Protean TGX 4-20% SDS-PAGE gel is a pre-cast polyacrylamide gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins. The gel has a linear gradient of 4% to 20% polyacrylamide concentration, allowing for the separation of a wide range of molecular weight proteins.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc touch gel imaging system, by Bio-Rad (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Odyssey blot imager, by LI COR (2 mentions)
Nitroceullose membranes, by Bio-Rad (2 mentions)
Trans blot turbo, by LI COR (2 mentions)
Intercept buffer, by LI COR (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Complete mini, by Roche (1 mentions)
Hgf 100 39, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Rabbit anti ucp1, by Abcam (1 mentions)
Rabbit anti β tubulin, by Cell Signaling Technology (1 mentions)
Mouse anti flag tag, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
6 protocols using mini protean tgx 4 20 sds page gel
1
Immunoprecipitation of E2F3 and RB
2
HGF-Induced Immunoblot Analysis
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Immunoblot was carried out as described in reference 14 (link). DAOY cells were prepared in six well plates to reach 70% confluency within 24 h. Standard media were replaced with SFM. After 24 h at 85% confluency, cells were treated with either SFM or SFM supplemented with 20 ng/ml HGF (100-39; PeproTech) for 30 min. Cells lysis was performed with RIPA buffer for 15 min on ice and cleared lysates were analyzed by SDS–PAGE. Protein concentration was assessed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Proteins were separated on Mini-Protean TGX (4-20%) SDS–PAGE gel (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). After 1 h of blocking with 5% non-fat milk, membranes were probed with primary antibodies listed in Supplementary material (Table S7). GAPDH was used as the internal loading control. HRP-linked secondary antibodies (1:2,000) were used to detect the primary antibodies. Chemiluminescence detection was carried out using ChemiDoc Touch Gel imaging system (Bio-Rad). The integrated density of detected bands was quantified using Image Lab software (Bio-Rad).
3
HGF-Induced Signaling Pathway Analysis
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Immunoblot was carried out as described in 14 . DAOY cells were prepared in 6 well plates to reach 70% confluency within 24 h. Standard media were replaced with SFM. After 24 h at 85% confluency, cells were treated with either SFM or SFM supplemented with 20 ng/ml HGF (PeproTech, 100-39) for 30 min. Cells lysis was performed with RIPA buffer for 15 min on ice and cleared lysates were analyzed by SDS-PAGE. Protein concentration was assessed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Proteins were separated on Mini-Protean TGX (4-20%) SDS-PAGE gel (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). Following 1 h of blocking with 5% non-fat milk, membranes were probed with primary antibodies listed in Supplementary material (Table S3). GAPDH was used as the internal loading control. HRP-linked secondary antibodies (1:2000) were used to detect the primary antibodies. Chemiluminescence detection was carried out using ChemiDoc Touch Gel imaging system (Bio-Rad). The integrated density of detected bands was quantified using Image Lab software (Bio-Rad).
4
Western Blotting Protocol for Protein Analysis
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Equal amounts of lysates were loaded onto Mini-Protean TGX (4-20%) SDS-PAGE gels (BioRad). All blots except phospho-MTFR1L were transferred to nitroceullose membranes (BioRad) with a Trans-Blot Turbo and blocked for one hour in Intercept Buffer (Li-COR). Membranes were then incubated in their respective primary antibodies in blocking buffer at 4°C overnight. Blots probing for phospho-MTFR1L we transferred to polyvinylidene difluoride membranes (PVDF, Immobilion) and blocked in 5% fat-free dry milk in TBS-T for 1 hour before primary antibody incubation for four days at 4°C. Membranes were incubated in Li-Cor fluorescence-coupled secondary antibodies for 1 hour at room temperature prior to visualization with a Li-Cor Odyssey Blot Imager.
5
Comprehensive Protein Extraction and Analysis
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Tissue fragments were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS using ball bearings and a TissueLyser (Qiagen). Lysis buffer was supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher). Protein lysates were quantitated using Pierce BCA protein assay (Thermo Fisher). Western blotting was performed using Miniprotean TGx 4–20% SDS-PAGE gels, Tetra Cell rig, and Transblot Turbo transfer system (Biorad). Membranes were blocked with 5% milk in 1× TBS-T, before overnight incubation with primary antibodies. HRP-conjugated secondary antibodies against rabbit- and mouse-derived primary antibodies were used at 5000-fold dilution and were obtained from Cell Signaling Technologies (7074S, 7076S). Chemiluminescence was visualized and recorded digitally using the Chemidoc XRS + Imaging System (Biorad). Primary antibodies used in this study include: rabbit anti-Cdkal1 (abcam, ab68045), mouse anti-ANT1 (abcam, 110322), rabbit anti-MMS19 (Proteintech, 16015-1-AP), mouse anti-total OXPHOS rodent cocktail (abcam, ab110413), mouse anti-FAM96B (Santa Cruz, sc-376801), mouse anti-FLAG tag (Sigma, F1804), mouse anti-FLAG M2 HRP-conjugated (Sigma, A8592), rabbit anti-UCP1 (abcam, 23841), and rabbit anti-β-Tubulin (Cell Signaling, 2146).
6
Western Blot Protocol
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Equal amounts of lysates were loaded onto Mini-Protean TGX (4-20%) SDS-PAGE gels (BioRad). All blots except phospho-MTFR1L were transferred to nitroceullose membranes (BioRad) with a Trans-Blot Turbo and blocked for one hour in Intercept Buffer (Li-COR). Membranes were then incubated in their respective primary antibodies in blocking buffer at 4 °C overnight. Blots probing for phospho-MTFR1L we transferred to polyvinylidene difluoride membranes (PVDF, Immobilion) and blocked in 5% fat-free dry milk in TBS-T for 1 hour before primary antibody incubation for four days at 4 °C. Membranes were incubated in Li-Cor fluorescence-coupled secondary antibodies for 1 hour at room temperature prior to visualization with a Li-Cor Odyssey Blot Imager.
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