Lung tissues were homogenized and protein was isolated using the RIPA lysis solution (P0013B, Beyotime, Shanghai, China). Protein concentration was determined with a Bradford protein assay kit (P0009, Beyotime). A total of 15–30 µg protein per lane was separated on 10% SDS polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). Following blocking in TBST (TBS with 0.1% Tween-20) for 1 h, the membrane was incubated with anti-S1PR1 (1:500; A12935, ABclonal, Wuhan, China), anti-nuclear factor kappa-B (NF-κB) ligand p65 (1:1,000; A19653, ABclonal), anti-phospho-inhibitor of NF-κB (p-IκB)-α (Ser32/36; 1:500; AF2002, Affinity Biosciences, Changzhou, China), anti-IκB-α (1:1,000; AF5002, Affinity), anti-p-IκB kinase (IKK; Ser180/Ser181; 1:500; AF3013, Affinity, China), anti-IKK (1:1,000; AF6014, Affinity) and anti-Histone H3 (1:500; 17168-1-AP, Proteintech, Wuhan, China), and anti-β-actin (1:2,000; 60008-1-Ig, Proteintech) antibodies at 4°C overnight. Membranes were incubated with the secondary antibodies at 37°C for 40 min. The relative intensity of proteins was visualized using electrochemiluminescence reagents (Shanghai 7sea biotech Co., Ltd.) and quantified using Gel-Pro Analyzer 4.0 (Media Cybernetics, Rockville, MD, USA).
Af3013
Manufactured by Affinity Biosciences
Sourced in China
The AF3013 is a laboratory centrifuge designed for general-purpose applications. It features a compact and durable construction, and can accommodate a variety of rotor types to suit different sample volumes and tube sizes. The centrifuge provides consistent and reliable performance, enabling efficient separation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Af2002, by Affinity Biosciences (3 mentions)
Necrostatin 1, by Selleck Chemicals (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Af5002, by Affinity Biosciences (1 mentions)
A19653, by ABclonal (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Ab73400, by Abcam (1 mentions)
Ab214821, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Ab32539, by Abcam (1 mentions)
Ab2796, by Abcam (1 mentions)
Ab32041, by Abcam (1 mentions)
Ab207297, by Abcam (1 mentions)
Ab45039, by Abcam (1 mentions)
Ab110304, by Abcam (1 mentions)
Af2006, by Affinity Biosciences (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab19027, by Abcam (1 mentions)
3 protocols using af3013
1
Quantification of Protein Expression in Lung Tissues
2
Western Blot Analysis of Osteoblast Markers
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The total proteins of MC3T3-E1 cells or BMSCs were extracted and lysed with RIPA buffer (Beyotime, China), and protein concentration was detected using a BCA protein assay kit (Solarbio, China). Then, a 20 μg total protein sample was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred to a PVDF membrane. After being blocked with 5% skimmed milk, the membrane was incubated at 37°C with the primary antibodies against caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), SIRT1 (ab110304, Abcam), BMP-2 (ab214821, Abcam), RANKL (ab45039, Abcam), OPG (ab73400, Abcam), IκBα (ab32518, Abcam), p-IκBα (AF2002, Affinity), p65 (ab207297, Abcam), p-p65 (AF2006, Affinity), IKKα (ab32041, Abcam), p-IKKα (AF3013, Affinity), NFATc1 (ab2796, Abcam), and cathepsin K (ab19027, Abcam) at 4°C overnight. After that, the membrane was further incubated with the secondary antibody at room temperature for 1 h. Then, the enhanced chemiluminescence (ECL) reagent (Solarbio, China) was used to visualize the protein bands, and the relative band density was semiquantified with the ImageJ software.
3
Investigating Necroptosis Signaling Pathways
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The following commercial antibodies and reagents were used: Necrostatin-1 (S8037, Selleck); Necrostatin 1S (S8641, Selleck); Z-VAD-FMK (S0723, Selleck); TPCA-1(S2824, Selleck); PH-797804 (S2726, Selleck); SP600125 (S1460, Selleck); C-176 (S6575, Selleck); QNZ (S4902, Selleck); RIPK1 (1:1000, 551041, BD Biosciences); RIPK3 (1:1000, NBP1-77299, NOVUS); MLKL (1:2000, AP14272B, Abcepta); pMLKL (1:1000, 37333S, Cell Signaling Technology); Flag (1:1000, 20543-1-AP, Proteintech); caspase3 (1:1000, 19677-1-AP, Proteintech); HMGB1 (1:1000, 10829-1-AP, Proteintech); p65 (1:1000, 66535-1-Ig, Proteintech); GAPDH (1:10,000, 60004-1-Ig, Proteintech); Histone H3 (1:1000, 17168-1-AP, Proteintech);IL6 (1:1000, 66146-1-Ig, Proteintech); CD68 (1:1000, 28058-1-AP, Proteintech); TMEM119 (1:1000, 27585-1-AP, Proteintech); p-p65 (1:200, sc-136548, Santa Cruz); IKK (1:500, AF6009, Affinity); p-IKK (1:500, AF3013, Affinity); IKbα (1:1000, 10268-1-AP, Proteintech); p-IKbα (1:500, AF2002, Affinity); Ccl5 (1:500, AF5151, Affinity); IFNb1 (1:500, DF6471, Affinity); pTau396 (1:1000, 829,001, Biolegend); AT8 (1:1000, MN1020, Thermo); Goat Anti-Mouse-HRP IgG (1:10,000, 115-035-003, Jackson ImmunoResearch); Goat Anti-Rabbit-TRITC IgG (1:200, 111-025-045, Jackson ImmunoResearch); Goat Anti-Rabbit-HRP IgG (1:10,000, 111-005-003, Jackson ImmunoResearch).
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