Cells were washed twice in PBS, collected, and lysed in cell lysis buffer on ice for 30 min and then sonicated for 1 min at 60 of amplitude with 2 s intervals. After centrifugation at 10,000g, 4 °C for 10 min, supernatant was transferred into new tubes. The concentration of the protein sample was measured by bicinchoninic acid, and then protein samples were boiled in SDS Sample Buffer at 99 °C for 10 min. 20 μg or 40 μg (for Tcstv1/3 and Zscan4) total proteins of each cell extracts were resolved by 10% or 12% (for Tcstv1/3) Bis-Tris SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Millipore). Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 2 h. Blots were then probed with primary antibodies, Tcstv1/3 (custom-made), Zscan4 (AB4340; Millipore), H3K9me3(ab8898; Abcam), H3K9Ac (04-1003; Millipore), H3Ac (06-599; Millipore), H3 (ab1791; Abcam), Dnmt3a (ab13888; Abcam), Dnmt3b (ab13604; Abcam) and β-actin (P30002; Abmart) by overnight incubation at 4 °C in 5% skim milk in TBST. Immunoreactive bands were then probed for 2 h at room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-Rabbit IgG-HRP (GE Healthcare, NA934V), or goat anti-Mouse IgG (H + L)/HRP (ZSGB-BIO, ZB-2305). Protein bands were detected by Chemiluminescent HRP substrate (Millipore, WBKLS0500).
P30002
Manufactured by Abmart
Sourced in China, United States
The P30002 is a versatile laboratory equipment designed for a variety of scientific applications. It features a compact and durable construction, ensuring reliable performance in the laboratory environment. The core function of the P30002 is to provide precise and consistent measurements, making it a valuable tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Ab13604, by Abcam (2 mentions)
Ab8898, by Abcam (2 mentions)
Ab13888, by Abcam (2 mentions)
Ab4340, by Merck Group (2 mentions)
Ab1791, by Abcam (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Wbkls0500, by Merck Group (1 mentions)
Ab37150, by Abcam (1 mentions)
Ab53519, by Abcam (1 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (1 mentions)
Ab33168, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sc 2005, by Santa Cruz Biotechnology (1 mentions)
Sc 2030, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Sc 11769, by Santa Cruz Biotechnology (1 mentions)
Sc 8426, by Santa Cruz Biotechnology (1 mentions)
Sc 15393, by Santa Cruz Biotechnology (1 mentions)
Sc 924, by Santa Cruz Biotechnology (1 mentions)
7 protocols using p30002
1
Western Blot Analysis of Epigenetic Markers
2
Western Blot Analysis of EMT Markers
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Cells (please see below) were lysed by using lysis buffer for western blotting (P00l3, Beyotime) and loaded onto 10% sodium dodecyl sulphate-polyacrylamide gels and were then transferred onto polyvinylidene difluoride membranes (Millipore). The quantification of total protein was performed using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific), and equal amounts of protein (30 μg) were used for analysis. Blots were blocked with 5% milk/TBST and incubated with primary antibodies (Twist1: 1:500, sc-15393, Santa Cruz Biotechnology; Slug: 1:1,000, 6591, Cell Signaling Technology; Snail: 1:1,000, ab53519, Abcam; Vimentin: 1:1,000, 2707-1, Epitomis; EphA2: 1:500, sc-924, Santa Cruz Biotechnology; VE-cadherin: 1:500, ab33168, Abcam; MMP2: 1:500, ab37150, Abcam; E-cadherin: 1:200, SC-8426, Santa Cruz Biotechnology; p-smad2/3: 1:500, sc-11769, Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with secondary antibodies (goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP, 1:2,000; sc-2005 and sc-2030; Santa Cruz Biotechnology) for 2 h at room temperature. Blots were developed using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech). For protein loading analyses, p-actin antibody (1:1,000, P30002, Abmart) or GAPDH (1:2,000, ab9485, Abcam) was used.
3
Western Blot Analysis of HIF-1α and Sema4D
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Cells were lysed in RIPA buffer (R0010, Solarbio, Beijing, China) with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, Sigma, USA) for 20 min at 4 °C and collected in 1.5 mL Eppendorf tubes, followed by centrifugation (14000 g, 10 min). The precipitate was discarded and protein concentrations were measured using the BCA Protein Assay Kit (P0010S, Beyotime biotechnology, Beijing, China). Forty micrograms of protein from each sample were subjected to SDS-PAGE followed by transfer onto a PVDF membrane (IPVH00010, Immobilon P, Millipore, Bedford, MA). The membrane was then probed for HIF-1α, Sema4D, using β-actin as a loading control. The primary antibodies used were as follows: rabbit anti-Sema4D (610670, BD Biosciences, 150 kDa); mouse anti-HIF-1α (D43B5, Cell Signaling Technology, 120 kDa), rabbit anti-β-actin (P30002, Abmart, 42 kDa). The secondary antibodies used were as follows: goat anti-mouse IgG (H+L) peroxidase labeled (074-1806, KPL, MD, USA), goat anti-rabbit IgG (H+L) peroxidase labeled (074-1506, KPL, MD, USA). Antigen-antibody binding was detected using Luminata Crescendo Western HRP substrate (WBLUR0100, Millipore, MA, USA).
4
Protein Expression Analysis of Stem Cell Markers
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Cells were washed twice in PBS, collected, lysed, and boiled in SDS sample buffer at 99 °C for 5 min; Equal amounts of total proteins of each cell extracts were resolved by 10–12% Bis-Tris SDS-PAGE and transferred to polyvinylidinedifluoride membranes (PVDF, Millipore, Burlington, MA, USA). Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 1–2 h. Blots were then probed with various primary antibodies, anti-Zscan4 (AB4340, Millipore, 1:1000), Dnmt1 (sc10221, Santa Cruz, 1:500), Dnmt3a (ab13888, Abcam, 1:1000), Dnmt3b (ab13604, Abcam, 1:1000), Tet2 (Kind gift from Dr. Jinsong Li from SIBS, 1:1000), H3K9me3 (ab8898, Abcam, 1:2000), H3 (ab1791, Abcam, 1:2000), and β-actin ((P30002, Abmart, 1:5000) by overnight incubation in 5% skim milk in TBST at 4 °C. Immunoreactive bands were then probed for 1–2 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary anti-Rabbit IgG-HRP (GE Healthcare, NA934V, 1:5000), or anti-mouse IgG-HRP (Santa Cruz, sc-2031, 1:5000), or anti-goat IgG-HRP (Santa Cruz, sc-2020, 1:5000). The protein bands were detected by Enhanced ECL AmershamTM prime Western blotting detection reagent (GE Healthcare, RPN2232).
5
Western Blot Analysis of Protein Expression in BMSCs
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Total cell protein was extracted from BMSCs according to the SAB Total Protein Extraction Kit (SAB, USA) kit instructions from BMSCs. A BCA (Beyotime, China) assay kit was used to measure the protein concentration. 30 micrograms of protein from each sample was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with skim milk, the membranes were incubated with the following primary antibodies overnight at 4℃: Bmal1 (ABCAm, ab93806), GAPDH (CST, 2118 S), YAP (ABCAm, ab205270), p-YAP (ABCAm, ab76252), TAZ (ABCAm, ab84927), MST1 (CST, 3682 S), MST2 (CST, 3952 S), P-MST1/2 (CST, 49,332 S), LATS1 (ABCAm, ab70561), LATS2 (ABCAm, ab135794), ALP (Affinity, DF12525), RUNX2 (Bioss, bs-1134R), OCN (Affinity, DF12303), P53 (ABCAm, ab26), P16 (ABCAm, ab80) and β-actin (Abmart, P30002). The antibodies were diluted in Tris-buffered saline (TBS) with Tween-20 (TBST) at 1:1000. Subsequently, the membranes were immersed in HRP universal secondary antibody (1:2000, CST, 7074,) for 1 h. Afterwards, the protein bands were visualized according to the ECL kit (Millipore, USA), and the gray values were analyzed using ImageJ software.
6
Western Blot Analysis of Protein Markers
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Western blot analysis was performed as previously described [17 (link)]. Primary antibodies included anti-GAPDH (1:7000, 10494–1-AP, Proteintech, China), anti-β-actin (1:8000, P30002, Abmart), anti-FXR (1:1000, 25055–1-AP, Proteintech, China), anti-caspase-1 (1:1000, 22915–1-AP, Proteintech, China), anti-IL-1β (1:1000, 16806–1-AP, Proteintech, China), anti-SMA (1:1000, 14395–1-AP, Proteintech, China), anti-ASC (1:500, 10500–1-AP, Proteintech, China), anti-collagen I (1:1000, ab260043, Abcam), anti-phospho-NLRP3 (Ser 295) (1:500, TA4320, Abmart) and anti-NLRP3 (1:1000, ab263899, Abcam). The secondary antibodies included HRP-conjugated Affinipure goat anti-rabbit IgG (H + L) (1:7000, SA00001-2, Proteintech, China) and HRP-conjugated Affinipure goat anti-mouse IgG (H + L) (1:7000, SA00001-1, Proteintech, China).
7
Immunoblotting and Immunofluorescence of Fetal Tissues
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Western blotting of craniofacial and spinal column tissue homogenates from fetuses was performed according to standard protocols. The primary antibodies that were used were rabbit anti-LOXL3 (1:400, 20–5193; American Research Products, Inc., Waltham, USA) and rabbit anti-β-actin antibodies (1:5000, P30002, Abmart). The secondary antibody that were used was goat anti-rabbit IgG-HRP (1:5000, ZB-2301; ZSGB-BIO). We preformed immunofluorescence staining on sagittal sections of the spinal columns and coronal sections from the anterior region of the palates from fetuses. The primary antibody that was used was rabbit anti-LOXL3 (1:400, 20–5193; American Research Products, Inc., Waltham, USA).The secondary antibody that was used was fluorescein-conjugated affinipure goat anti-rabbit IgG (H+L) (1:200; ZF-0311; ZSGB-BIO).
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