Real-time PCR analysis was performed as previously described with primers and probe sets from Applied Biosystems31 (link). Immunoblots were performed and quantified as described previously12 (link), using the following antibodies: NF-κB2 (#4882), RelB (#4954), p-Mst1/2 (#3681), p-Yap (Ser127) (D9W2I), Lats1 (C66B5), Mst1 (D8B9Q), Mst2 (#3952), Actin (8H10D10), p-DRP1 (Ser637) (#4867), p-S6 (2F9), c-Myc (#9402s) (all from Cell Signaling Technology), OPA1 (NB110-55290SS, Novus Biologicals) and MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail (MS604, MitoSciences).
Actin 8h10d10
Manufactured by Cell Signaling Technology
Sourced in United States
Actin (8H10D10) is a monoclonal antibody that specifically recognizes actin, a highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used to detect and quantify actin in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab133612, by Abcam (2 mentions)
Ab209484, by Abcam (2 mentions)
Anti alp ab83259, by Abcam (2 mentions)
P mst1 2, by Cell Signaling Technology (1 mentions)
Nf κb2, by Cell Signaling Technology (1 mentions)
P drp1 ser637, by Cell Signaling Technology (1 mentions)
Mitoprofile total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
Pi cocktail, by Thermo Fisher Scientific (1 mentions)
Np 40 buffer, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Transfer buffer, by Thermo Fisher Scientific (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence kit, by GE Healthcare (1 mentions)
Goat anti mouse secondary antibody, by Merck Group (1 mentions)
6 protocols using actin 8h10d10
1
Comprehensive Molecular Profiling of Cellular Signaling
2
Protein Lysate Isolation and Western Blot
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Protein lysates were isolated from purified B cells, platelets or in vitro generated MKs with NP‐40 buffer (Thermo Scientific) containing 1 mmol/L PMSF (Sigma‐Aldrich) and PI cocktail (Thermo Scientific) for 30 minutes on ice followed by 10 minutes centrifugation at 15 000 g. The supernatant was used for SDS‐PAGE followed by Western blot analysis. Antibodies for c‐CBL (#2747, 1:1000), Actin (8H10D10, 1:5000) (all from Cell Signaling) and c‐MPL (#06‐944; Merck) and horseradish peroxidase‐coupled goat or swine anti‐rabbit secondary antibody (Southern Biotech) were used.
3
PTPN22 Expression in Edited T Cells
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All western blots were performed on lysates from human umbilical cord blood-derived CD4 T cells that had been either mock edited or gene edited, expanded 7 days in cytokine supplemented media, and subjected to 24 hr rest in cytokine free media. Cells were lysed in 1× RIPA lysis buffer on ice for 10 min then clarified by centrifugation. Concentration of clarified lysate was determined by BCA assay (Pierce), diluted, and suspended in 1× LDS Sample Buffer (Invitrogen). Ten μg of lysate was run on 4–12% Bis-Tris NuPAGE gels in 1× MOPS buffer (Invitrogen). Protein was transferred to nitrocellulose in 1× Transfer Buffer (Invitrogen) and 10% methanol. Non-specific binding was minimized with a 1 hr room temperature (RT) incubation in Odyssey LI-COR Blocking Buffer. Primary antibodies were stained at 1:1000. PTPN22 primary stain was for at least 12 hr at 4°C and actin was stained at RT for 40 min. Primary antibodies used were from Cell Signaling Technology: PTPN22 (D6D1H, Cat# 3700, RRID:AB_2798575 ) and actin (8H10D10, Cat# 14693, RRID:AB_2242334 ). After primary stain membranes were washed with 1× TBST and incubated with secondary antibodies at 1:10,000 for 30 min at RT. Stained blots were washed and imaged on an Odyssey Infrared Imaging System (LI-COR Biotech.). Western blot quantifications were performed with ImageJ software.
4
Western Blot Analysis of Alzheimer's Biomarkers
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For western blot analysis, 60 μg of protein was fractioned on 12% SDS-PAGE and transferred to PVDF membranes. Following the transfer, samples were blocked with 5% nonfat milk in PBS containing 10% Tween-20 for 1 h and probed with primary monoclonal antibodies against Aβ1-16 (6E10, Abcam, 1:2000 dilution), Actin (8H10D10, Cell Signaling, 1:5000), PI3K (p85, Millipore, 1:1000) and phospho-PI3K (p85/phospho-Y607, Abcam, 1:500), Akt (11E7, Cell Signaling, 1:1000) and phospho-Akt (PKBα(Ser473), Millipore, 1:500), or Bad (D24A9, 1:200) and phospho-Bad (40A9, phosphor-Ser112, both Cell Signaling, 1:1000) overnight at 4 °C. After probing with primary antibodies, the PVDF membrane was washed three times with PBST and probed with goat anti-mouse secondary antibody (Sigma, 1:5000 dilution), then detected by using a chemiluminescence kit (GE, Pittsburgh, PA, USA). Following the manufacturer’s protocol, the detection solution was prepared by mixing a 1:1 ratio of reagent A (luminol chemicals) and B (peroxide solution). The sample blots were incubated with the detection solution for 5 min and then detected using a CCD camera image system (UVP, Rockland Immunochemical Inc., Limerick, PA, USA). The blot images were analyzed using the ImageJ program.
5
In Vivo Bone Formation Assay
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Western blot analysis was performed as previously reported (24) . The primary antibodies were as follows:
anti-ALP (ab83259) (1:1000), anti-osteocalcin (OCN) (ab133612) (1:1000), and anti-Osterix (OSX) (ab209484) (1:1000) (all from Abcam, MA, USA; and RUNX2 (D1L7F) rabbit mAb #12556 (1:1000), APC antibody #2504 (1:1000), 𝛽-catenin (D10A8) XP Rabbit mAb #8480 (1:1000), and 𝛽-actin (8H10D10) mouse mAb #3700 (1:1000) (all from Cell Signaling Technology MA, USA). 𝛽-Actin served as an internal control. Relative densitometry analysis of the Western blot was carried out using ImageJ software.
Relative protein levels were quanti ed as the ratio of the level of target protein to the level of 𝛽-actin, in each group.
In vivo bone formation assay Approximately 10 5 cells (5 × 10 4 HAMSCs and 5 × 10 4 HBMSCs NC /HBMSCs ANRIL / HBMSCs shNC /HBMSCs shANRIL pretreated with LPS) were attached to each HA/TCP biomaterial (Φ 5 × H 2 mm, Sichuan University, Chengdu, Sichuan, China). After 12 hours, the complexes were subcutaneously implanted into the rat mandibular defect area designed as previously reported (four female nude rats per group, with an average weight of 280 g) (23) . All animal experiments were conducted in compliance with the regulations and guidelines of Nanjing Medical University institutional animal care.
anti-ALP (ab83259) (1:1000), anti-osteocalcin (OCN) (ab133612) (1:1000), and anti-Osterix (OSX) (ab209484) (1:1000) (all from Abcam, MA, USA; and RUNX2 (D1L7F) rabbit mAb #12556 (1:1000), APC antibody #2504 (1:1000), 𝛽-catenin (D10A8) XP Rabbit mAb #8480 (1:1000), and 𝛽-actin (8H10D10) mouse mAb #3700 (1:1000) (all from Cell Signaling Technology MA, USA). 𝛽-Actin served as an internal control. Relative densitometry analysis of the Western blot was carried out using ImageJ software.
Relative protein levels were quanti ed as the ratio of the level of target protein to the level of 𝛽-actin, in each group.
In vivo bone formation assay Approximately 10 5 cells (5 × 10 4 HAMSCs and 5 × 10 4 HBMSCs NC /HBMSCs ANRIL / HBMSCs shNC /HBMSCs shANRIL pretreated with LPS) were attached to each HA/TCP biomaterial (Φ 5 × H 2 mm, Sichuan University, Chengdu, Sichuan, China). After 12 hours, the complexes were subcutaneously implanted into the rat mandibular defect area designed as previously reported (four female nude rats per group, with an average weight of 280 g) (23) . All animal experiments were conducted in compliance with the regulations and guidelines of Nanjing Medical University institutional animal care.
6
In Vivo Bone Formation Assay
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Western blot analysis was performed as previously reported (24) . The primary antibodies were as follows:
anti-ALP (ab83259) (1:1000), anti-osteocalcin (OCN) (ab133612) (1:1000), and anti-Osterix (OSX) (ab209484) (1:1000) (all from Abcam, MA, USA; and RUNX2 (D1L7F) rabbit mAb #12556 (1:1000), APC antibody #2504 (1:1000), 𝛽-catenin (D10A8) XP Rabbit mAb #8480 (1:1000), and 𝛽-actin (8H10D10) mouse mAb #3700 (1:1000) (all from Cell Signaling Technology MA, USA). 𝛽-Actin served as an internal control. Relative densitometry analysis of the Western blot was carried out using ImageJ software.
Relative protein levels were quanti ed as the ratio of the level of target protein to the level of 𝛽-actin, in each group.
In vivo bone formation assay Approximately 10 5 cells (5 × 10 4 HAMSCs and 5 × 10 4 HBMSCs NC /HBMSCs ANRIL / HBMSCs shNC /HBMSCs shANRIL pretreated with LPS) were attached to each HA/TCP biomaterial (Φ 5 × H 2 mm, Sichuan University, Chengdu, Sichuan, China). After 12 hours, the complexes were subcutaneously implanted into the rat mandibular defect area designed as previously reported (four female nude rats per group, with an average weight of 280 g) (23) . All animal experiments were conducted in compliance with the regulations and guidelines of Nanjing Medical University institutional animal care.
anti-ALP (ab83259) (1:1000), anti-osteocalcin (OCN) (ab133612) (1:1000), and anti-Osterix (OSX) (ab209484) (1:1000) (all from Abcam, MA, USA; and RUNX2 (D1L7F) rabbit mAb #12556 (1:1000), APC antibody #2504 (1:1000), 𝛽-catenin (D10A8) XP Rabbit mAb #8480 (1:1000), and 𝛽-actin (8H10D10) mouse mAb #3700 (1:1000) (all from Cell Signaling Technology MA, USA). 𝛽-Actin served as an internal control. Relative densitometry analysis of the Western blot was carried out using ImageJ software.
Relative protein levels were quanti ed as the ratio of the level of target protein to the level of 𝛽-actin, in each group.
In vivo bone formation assay Approximately 10 5 cells (5 × 10 4 HAMSCs and 5 × 10 4 HBMSCs NC /HBMSCs ANRIL / HBMSCs shNC /HBMSCs shANRIL pretreated with LPS) were attached to each HA/TCP biomaterial (Φ 5 × H 2 mm, Sichuan University, Chengdu, Sichuan, China). After 12 hours, the complexes were subcutaneously implanted into the rat mandibular defect area designed as previously reported (four female nude rats per group, with an average weight of 280 g) (23) . All animal experiments were conducted in compliance with the regulations and guidelines of Nanjing Medical University institutional animal care.
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