Protein samples were extracted by using NP40 solution followed manufactory’s protocol (Thermofisher) and protein concentration was determined by using Qubit protein detection Kit (Invitrogen) followed by manufactory’s protocol. Individual samples were run on a single 10 well gel or 12 well pre-made 4–12% gel (Thermofisher). Briefly, samples were prepared on ice (to final volume of 20 μl) and then vortexed and denatured for 10 min at 90 °C. Gels were run with 1X Tris buffered saline-Tween (TBS-T) and proteins transferred onto nitrocellulose membrane (Bio-rad). The membrane was blocked by blocking buffer (Licor) for 1hr at room temperature, washed with TBS-T for 5 min (3X), and incubated with 5ml of N6amt1 (1:250; Santa Cruz) and Beta-actin (1:500; Santa Cruz) or beta-tubulin (1:500; Santa Cruz) antibodies in blocking buffer (Licor) for overnight at 4 °C. The membranes were washed with TBS-T (3X), incubated for 1hr with anti-mouse secondary antibody (1: 15000; Li-Cor) and anti-rabbit secondary antibody (1:15000; Li-Cor) in blocking buffer (Li-Cor), and washed three times with TBST for 10 min (5X) and 20 min (1X). Optical density readings of the membrane were taken using a Li-Cor FX system followed by manufactory’s protocol. Detailed antibody information is attached within Reporting Summary file.
Anti rabbit secondary antibody
Manufactured by LI COR
Sourced in Germany
The anti-rabbit secondary antibody is a laboratory reagent used to detect the presence of primary antibodies that target rabbit-derived proteins. It binds to the Fc region of rabbit primary antibodies, allowing for their identification and quantification in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Np40 solution, by Thermo Fisher Scientific (4 mentions)
Qubit protein detection kit, by Thermo Fisher Scientific (4 mentions)
Fx system, by LI COR (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
N6amt1, by Santa Cruz Biotechnology (2 mentions)
β tubulin, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ab11174, by Evrogen (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Ab961, by Evrogen (1 mentions)
Spin x column, by Corning (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imaging device, by LI COR (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Ab76020, by Abcam (1 mentions)
13 protocols using anti rabbit secondary antibody
1
Western Blot Protein Detection
2
Western Blot Protein Detection
3
Western Blot Analysis of γH2AX and KillerRed Proteins
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293T cells with or without the dCas9-KR and sgAAVS1 constructs were lysed in RIPA buffer (50mM Tris-HCl, pH 8.0; 150mM sodium chloride; 1.0% NP-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate) with 1X protease inhibitor cocktail (Roche, IN) and sonicated in a bioruptor for 2.5 mins in 30 second pulses at 4°C. Debris was pelleted at 10,000 rpm and lysates (20 μg) separated in SDS-PAGE gels and semi-dry transferred onto nitrocellulose using standard methods. Primary antibodies (diluted in 5% milk) used were: anti-γH2AX (Abcam ab11174; 1:5000), anti-KillerRed (Evrogen AB961; 1:5000), and anti-β-actin (Cell Signaling Technologies 4967; 1:2000). Bands were detected with anti-rabbit secondary antibody (LI-COR; 1:10,000) and scanned on an Odyssey imager to quantify band intensity.
4
Immunoblotting Analysis of CinA Protein
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Protein extracts were prepared from wild-type, ΔcinA, ΔcinA:: cinA, ΔcinA:: cinAD80A, ΔcinA:: cinAK323A, cinA-TetON strains by mechanical lysis with 0.1 mm zirconia/silica beads in PBS in presence of protease inhibitor cocktail (Roche). Subsequently, lysates were filtered through 0.22 μm Spin-X columns (Corning). Protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad). For immunoblot analysis of CinA, 20 or 40 μg of protein was resolved through SDS-page, transferred to nitrocellulose membrane and probed with anti-CinA serum (1:50 dilution) and anti-PrcB (1:10,000 dilution) or anti-PckA (1:10,000 dilution). Following incubation with anti-rabbit secondary antibody (LI-COR Biosciences), protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).
5
Western Blot Protein Detection Protocol
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Whole-cell lysates (15 μg) were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using a semidry transfer system (iBlot 2; Invitrogen). The membrane was then blocked overnight with blocking solution containing 4% milk (dry milk dissolved in 20 mM Tris-HCl [pH 7.8], 150 mM NaCl, 0.1% Triton X-100). The next day, the membrane was incubated with anti-ShyA polyclonal antibody (1:5,000) (produced by Pocono Rabbit Farm & Laboratory, PA) for 2 h and then washed twice with 1× TBST (20 mM Tris-HCl [pH 7.8], 150 mM NaCl, 0.1% Triton X-100). The washed membranes were then incubated with anti-rabbit secondary antibody (1:15,000) (Li-Cor catalog no. 926-32211) for 1 h. Membranes were then washed three times with 1× TBST, scanned on an Odyssey CLx imaging device (Li-Cor Biosciences) and visualized using Image Studio Lite version 5.2 software (Li-Cor) for signal quantification.
6
Integrase Crosslinking Assay
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Peptide 15 (111 µM), containing a photo-reactive Bpa group and a biotin linked to its amino terminus, was incubated for 2 h at 37 °C in the absence or the presence of IN (3.2 µM) before UV irradiation (5 min at 254 nM). After adding 2 × 10 µL of loading buffer, samples were heat-denatured at 95 °C (5 min). Products were separated on 16% SDS-PAGE and transferred to a PVDF membrane using an iBlot system (Invitrogen, Carlsbad, CA, USA). IN was revealed by Western blot using a primary rabbit polyclonal antibody directed against the IN CTD (AIDS Reagent program, cat. No. 758) and an anti-rabbit secondary antibody from Li-COR (680 nm), while the biotinylated peptide was revealed using streptavidin coupled to AlexaFluor 750. Reading was performed using an Odyssey® Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA).
7
Na/K ATPase and FXYD3 Protein Analysis
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Samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-FL, Millipore). The total protein for each lane was detected with revert total protein stain and washed as per manufacturer’s protocol (LI-COR). Following total protein detection, the membrane was blocked with 0.1% casein protein in PBS for 1 h at room temperature. Then, the membrane was concurrently probed with rabbit Na/K ATPase (1:1,000 dilution; Abcam; ab76020; Lot GR3184452‐8) and rabbit FXYD3 (1:250 dilution, Sigma-Aldrich; HPA010856; Lot A57803) antibodies for 2 h at room temperature. Following PBS washes, the membrane was incubated with a 1:10,000 dilution of anti-rabbit secondary antibody (LI-COR). After a final PBS wash, images were obtained with an Odyssey gel imager (LI-COR), and raw signal intensities were analyzed with ImageJ (v2.3.0/1.53f, NIH) using the approach of the NIH ImageJ user guide. We used the area under both bands observed with the Na/K ATPase for analysis because heating the sample induces Na/K ATPase dimers (11 (link)). Total protein staining was the gel loading control, and data are presented as the %FXYD3 siRNA normalized to its donor-matched siRNA control.
8
Western Blot Analysis of Key Cellular Markers
9
Western Blot Protein Detection Protocol
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Protein samples were extracted using NP40 solution following the manufacturer’s protocol (Thermo Fisher) and the protein concentration was determined using the Qubit protein detection kit (Invitrogen), also following the manufacturer’s protocol. Individual samples were run on a single 10-well gel pre-made 4–12% gel (Thermo Fisher). Briefly, samples were prepared on ice (to a final volume of 20 μL) and then vortexed and denatured for 10 min at 90°C. Gels were run with TBS-T and proteins were transferred onto a nitrocellulose membrane (Biorad). The membrane was blocked by blocking buffer (LI-COR) for 1 h at room temperature, washed with TBS-T for 5 min (three times) and incubated with 5 mL of 14-3-3 β/α and β-tubulin (control) antibodies (Table S4 ) in blocking buffer (LI-COR) overnight at 4°C. The membranes were washed with TBS-T (three times), incubated for 1 h with anti-mouse secondary antibody (1:15,000; LI-COR) and anti-rabbit secondary antibody (1:15,000; LI-COR) in blocking buffer (LI-COR), then washed with TBS-T for 10 min (three times) and 20 min (once). Absorbance readings of the membrane were taken using a LI-COR FX system following the manufacturer’s protocol.
10
Western Blotting of Vascular Proteins
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Western blotting was performed according to standard protocols described previously (Citation15). Briefly, thoracic aortas and CD4 + T cells were homogenized using icecold RIPA lysis buffer. The proteins lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked using 8% fat-free milk in TBS (Tris-buffered saline) with 0.5% Tween-20 for 2 h at room temperature and then probed with primary antibodies against phosphorylated eNOS at Ser 1177 (1:500; Cell Signaling Technology, Danvers, MA, USA), t-eNOS (1:1000; Cell Signaling Technology), p-STAT3 (1:400, Abcam), t-STAT3 (1:400, Abcam), ROR-γt (1:400, Abcam) and GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at 4°C overnight. The anti-rabbit secondary antibody (1:10000, Li-Cor Bioscience, Bad Homburg, Germany) was incubated for 2 h at room temperature. The protein bands were detected by Odyssey Western Blot Detection System (LI-COR) and the band intensities were evaluated with ImageJ.
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