Nuclear and cytoplasmic proteins were extracted with nuclear and cytoplasmic extraction reagents kits (Pierce, Rockford, IL, USA) according to the manufacturer's protocol for Nrf2, Ho1, and Nqo1 detection. Protein concentrations were measured using BCA protein assay. Proteins were separated by 10% or 12% SDS-PAGE and then transferred onto PVDF membranes. The transferred membranes were blocked with TBS-T (10 mmol/L Tris-HC1, 150 mmol/L NaC1, 0.1% Tween-20) containing 5% skim milk for 1 h at room temperature. The membranes were washed in TBS-T (10 min × 3) and then the membranes were incubated overnight at 4°C with anti-Ho1 (Abcam, Cambridge, UK), anti-Nqo1 (Sigma, St. Louis, USA), anti-Nrf2 (Santa Cruz, CA, USA), anti-β-actin (Abmart, Shanghai, China), and anti-PCNA (Santa Cruz, CA, USA) in TBS-T. The membranes were washed three times again in TBS-T; the membranes were incubated with horseradish peroxidase conjugated to anti-rabbit IgG (Weiao Biotech Ltd, Shanghai, China) overnight at 4°C. The expression of targeted proteins was determined using Odyssey machine and optical density of band was analyzed by using Image J software.
Anti β actin
Manufactured by Abmart
Sourced in United States, China
Anti-β-actin is a laboratory reagent used in various research and analytical applications. It is an antibody that specifically binds to the beta-actin protein, which is a ubiquitous and highly conserved cytoskeletal protein found in eukaryotic cells. The core function of Anti-β-actin is to provide a tool for the detection and quantification of beta-actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Anti gapdh, by Abmart (2 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Anti nqo1, by Merck Group (1 mentions)
Anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Anti ho 1, by Abcam (1 mentions)
Mouse anti caspase 1 p20, by Adipogen (1 mentions)
Anti mouse il 1β, by R&D Systems (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Nigericin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Mcc950, by Selleck Chemicals (1 mentions)
Ultrapure lps, by InvivoGen (1 mentions)
Anti asc, by Santa Cruz Biotechnology (1 mentions)
Sc 22514 r, by Santa Cruz Biotechnology (1 mentions)
17 protocols using anti β actin
1
Protein Extraction and Western Blot Analysis
2
Inflammasome Activation Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSU, Nigericin, ATP, PMA (phorbol‐12‐myristate‐13‐acetate), poly A:T, insulin, and glucose were purchased from Sigma. TR and MCC950 were obtained from Selleck. The ultrapure LPS and Pam3CSK4 (tripalmitoylcysteinylseryltetralysinelipopeptide) were from Invivogen. Imject‐Alum was from Pierce Biochemicals. MitoTracker and MitoSOX were from Invitrogen. Protein G agarose was from Millipore. Anti‐β‐actin (1:5,000, P30002) and Anti‐DYKDDDDK‐Tag mAb were from Abmart. Anti‐human pro‐IL‐1β (1:1,000, 60136‐1‐Ig), anti‐TRPV2 (1:1,000, 15991‐1‐AP), and anti‐HPGDS (1:1,000, 22522‐1‐AP) were from Proteintech. Anti‐mouse IL‐1β (1:1,000, AF‐401‐NA) was from R&D Systems. Anti‐mouse caspase‐1 (p20) (1:1,000, AG‐20B‐0042) and anti‐NLRP3 (1:1,000, AG‐20B‐0014) were from Adipogen. Anti‐human caspase‐1(1:1,000, 2225) was from Cell Signaling. Anti‐ASC (1:500, sc‐22514‐R) and anti‐NEK7 (1:500, SC‐50756) were from Santa Cruz. Anti‐human cleaved IL‐1β (1:1,000, A5208206) was from Sangon Biotech. Anti‐Flag (1:2,000, F2555) or anti‐VSV (1:2,000, V4888) was from Sigma. Recombinant human NLRP3 was from Novus Biologicals. Salmonella is a gift from R.V. Bruggen.
3
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell lysates (80 μg) were harvested, separated and then transferred onto PVDF membranes as previously described.23 (link) The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature before the addition of 5 mL of primary antibody. The antibodies used in this study included anti-PD-L1 (1:500; Proteintech) and anti-β-actin (1:2000; Abmart). The membrane was then washed with PBS and incubated with rabbit anti-HRP-conjugated secondary antibody (1:2000; Abmart)) for 1 h at room temperature. The bands were visualized using a chemiluminescence reagent (Millipore, USA).
4
PD-L1 Regulation and Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sources of chemicals, antibodies, plasmids and other reagents were as follows:
BMS1166 were provided by Prof. Dawei Ma (Shanghai Institute of Organic Chemistry, Shanghai, China). Chloroquine (# c6628, Sigma-Aldrich), bortezomib (# S1013, Selleck), anti-PD-L1 Ab for blockade (# 29E.2A3, BioLegend), PD-L1/PD-1 inhibitor 1 (#S7911, Selleck), tunicamycin (# 654380, Sigma-Aldrich), cycloheximide (Sigma-Aldrich), TPA/PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich)
Anti-GAPDH (Shanghai Kangchen), anti-PD-L1 (# PA5-20343, Life), anti-PD-1 (# PA5-20350, Life), anti-PD-L1 (# 51296, CST), anti-mouse PD-L1 Ab (# ab80276, Abcam), anti-α-Tubulin (# SC-5286, Santa Cruz Biotechnology), anti-EGFR Ab (# sc-03-G, Santa Cruz Biotechnology), anti-IGF1Rβ Ab (# 9750, CST), anti-Axl (# 4566, CST), anti-GM130 (# ab52649, abcam), anti-Bip (# 3177, CST), anti-CHOP (# 2895, CST), anti-β-actin (# P30002M, Abmart).
Plasmid: pCMV-hPD-L1, pCMV-hPD-1 and pCMV-mPD-L1-HA were purchased from Sino Biological Inc. pGL4.30-luc-NFAT (# E848A) was purchased from Promega. pCDH-PD-L1-WT, N200Q, N219Q, N192/219Q, N200/219Q or 3NQ-Flag were gifts from Prof. Mien-Chie Hung (The University of Texas, Houston, USA).
IFN-γ (Peprotech).
BMS1166 were provided by Prof. Dawei Ma (Shanghai Institute of Organic Chemistry, Shanghai, China). Chloroquine (# c6628, Sigma-Aldrich), bortezomib (# S1013, Selleck), anti-PD-L1 Ab for blockade (# 29E.2A3, BioLegend), PD-L1/PD-1 inhibitor 1 (#S7911, Selleck), tunicamycin (# 654380, Sigma-Aldrich), cycloheximide (Sigma-Aldrich), TPA/PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich)
Anti-GAPDH (Shanghai Kangchen), anti-PD-L1 (# PA5-20343, Life), anti-PD-1 (# PA5-20350, Life), anti-PD-L1 (# 51296, CST), anti-mouse PD-L1 Ab (# ab80276, Abcam), anti-α-Tubulin (# SC-5286, Santa Cruz Biotechnology), anti-EGFR Ab (# sc-03-G, Santa Cruz Biotechnology), anti-IGF1Rβ Ab (# 9750, CST), anti-Axl (# 4566, CST), anti-GM130 (# ab52649, abcam), anti-Bip (# 3177, CST), anti-CHOP (# 2895, CST), anti-β-actin (# P30002M, Abmart).
Plasmid: pCMV-hPD-L1, pCMV-hPD-1 and pCMV-mPD-L1-HA were purchased from Sino Biological Inc. pGL4.30-luc-NFAT (# E848A) was purchased from Promega. pCDH-PD-L1-WT, N200Q, N219Q, N192/219Q, N200/219Q or 3NQ-Flag were gifts from Prof. Mien-Chie Hung (The University of Texas, Houston, USA).
IFN-γ (Peprotech).
5
Immunoblotting Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in lysis buffer supplemented with protease and phosphatase inhibitor (MCE). Proteins were quantified by the Bradford assay (HyClone-Pierce). The proteins were then separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were probed with Abs overnight at 4 °C, and then incubated with a horseradish peroxidase-coupled secondary Ab. Detection was performed using a LumiGLO chemiluminescent substrate system. Anti-p-IGF-I receptor β (IRβ) Cell Signal Technology, USA anti-p-protein kinase B (AKT; 1:1000, Proteintech, China), anti-CAP1 (Abcam, USA), anti-resistin (1:1000, Abcam, USA), anti-p-vasodilator stimulated phosphoprotein (VASP; 1:1000; Cell Signal Technology, USA), anti-p-p65 (1:1000; Cell Signal Technology, USA), anti-Na+/K+-ATPase (1:1000; Cell Signal Technology, USA), anti-β-actin (1:2000, Abmart, USA), anti-GAPDH (1:2000, Abmart, USA) were used.
6
Comprehensive Characterization of Myelinating Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used: anti-MBP (rabbit 1:250) (Abcam, UK), anti-Sox10 (rabbit 1:200) (Abcam, UK), anti-MPZ (rabbit 1:250) (Abcam, UK), anti-GAP43 (rabbit 1:250) (Abcam, UK), anti-P75 NGF Receptor (rabbit 1:500, (Abcam, UK), anti-Neural Cell Adhesion Molecule (rabbit 1:500) (Millipore, USA),anti-S100 (rabbit 1:500) (DakoCytomation, USA), Krox20 (rabbit 1:100) (Covance, USA), anti-Sox2 (rabbit 1:200) (Epitomics,USA), anti-Oct6 (goat 1:100) (Santa Cruz, USA), anti-β-actin (Abmart, China). For immunohistochemistry and immunocytochemistry tests, the secondary antibodies were conjugated with Alexa 488 and Alexa 555 (goat and rabbit 1:500) (Invitrogen, USA).
7
Western Blot Analysis of Hep3B Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hep3B cells were lysed with Cell lysis buffer for Western and IP (Beyotime Biotechnology, Jiangsu, China) containing 1 % PMSF. The protein content of different fractions was detected via the bicinchoninic acid (BCA) method. The obtained protein samples were subjected to sodium dodecyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE) with 10% gel concentration, and transferred onto PVDF membranes with pre-cast gels using wet transfer with 250 mA for 1 h. Membranes were put in 5% skimmed milk and seal it for 2h, then the first antibody (anti-β-actin, 1:1000 PD-L1 (Abmart, Shanghai, China)), 1:1000 anti-STAT-1 (Abcam, MA, USA), anti-P-STAT1-1 (Abcam, USA), anti-IRF, Bcl-2 and Bad (Cell Signaling, USA) were incubated for 4° C overnight. The next day, membranes were probed with second antibody (HRP-anti-mouse/rabbit IgG Cat#7076, Cat#7074, antibody, CST, Beijing, China). After another three washes, signals were detected by LuminataTM Western HRP substrates (Millipore Corporation, Billerica, MA, USA) and Chemi-DocTM MP Imaging System (v5.2.1, Bio-Rad Laboratories, Inc, Hercules, CA, USA).
8
Exosomal Protein Profiling in SK-MEL-5 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein concentrations of exosomes derived from SK-MEL-5 cells (WT), PD-L1 knockout SK-MEL-5 cells (Pd-l1−/-), IFN-γ-treated SK-MEL-5 cells (WT+IFN-γ, Pd-l1−/-+IFN-γ) and PD-L1 overexpressing SK-MEL-5 cells (WT+PD-L1) were measured by BCA protein assay. Cells and exosomes pellets were lysed in radioimmunoprecipitation assay buffer (RIPA, CWBIO) and incubated at 4°C for 30 min. Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk for 2 h and incubated with primary antibodies overnight at 4°C. After washing with PBS, membranes were incubated with appropriate secondary antibodies at room temperature for 2 h. Protein bands were visualized with the HRP-enhanced chemiluminescence western blotting substrate (Advansta). The primary antibodies for western blot analysis are shown below: anti-PD-L1 (AbWays technology), anti-CD63 (ExoAb Antibody Kit, SBI), anti-CD81 (ExoAb Antibody Kit, SBI), anti-Alix (Santa cruza technology), anti-GAPDH (Abmart), anti-β-actin (Abmart). The secondary antibodies were included goat anti-rabbit (ExoAb Antibody Kit, SBI) and goat anti-mouse (Sera care).
9
Investigating Neu5Gc Uptake and Trafficking in Mammalian Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neu5Gc was purchased from Aladdin (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum, penicillin, streptomycin, phosphate-buffered saline (PBS), Hank’s Balanced Salt Solution (HBSS) were purchased from BI (Kibbutz Beit-Haemek, Israel). Mouse anti-ZO-1 antibody Alexa Flour 488 conjugate were purchased from Invitrogen (San Francisco, CA, USA). Anti-ZO-1 and occludin antibodies were purchased from Affinity (Changzhou, China). Anti-claudin-1, p65, p-p65, I-κBα, p-I-κBα, and anti-β-actin antibodies were purchased form Abmart (Shanghai, China). BAY 11-7082 was purchased from Beyotime (Shanghai, China). Fish skin gelatin, 1,2-diamino-4,5-methylene-dioxybenzene (DMB), and sodium azide was purchased from Sigma (St. Louis, MI, USA). Chlorpromazine hydrochloride, amiloride hydrochloride and the MTT Detection Kit were purchased from Solarbio (Beijing, China). Dynasore was purchased from MCE (Monmouth Junction, NJ, USA). Nocodazole and nystatin were purchased from Yuanye Bio-Tech (Shanghai, China). Monensin sodium salt and cytochalasin D were purchased from Aladdin (Shanghai, China). Bafilomycin A1 was purchased from LC Laboratories (Woburn, MA, USA). Brefeldin A was purchased from Selleckchem (Houston, TX, USA).
10
Western Blot Analysis of PI3K/AKT/mTOR Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue and cell lysates were prepared by strong radioimmunoprecipitation assay buffer (Beyotime, P0013B) containing protease inhibitors and supplemented with protein phosphatase inhibitors (mammalian cell entry [MCE]). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 6% skim milk in Tris-Buffered Saline Tween-20 for 1.5 h at room temperature and subsequently incubated with specific primary antibodies overnight at 4°C followed by incubation with secondary antibodies for 1 h. The primary antibodies used in this study were as follows: Anti-flag-tag (cell signaling technology [CST], 14793, 1:1000), anti-c-MYC (Abcam, ab32072, 1:1000), anti-β-Actin (Abmart, P30002, 1:4000), anti-PI3K (CST, 4292, 1:1000), anti-p-PI3K (CST, 4228, 1:1000), anti-AKT (CST, 2920, 1:2000), anti-p-AKT (CST, 4060, 1:2000), anti-mTOR (CST, 2983, 1:1000), anti-p-mTOR (CST, 5536, 1:1000), and anti-GAPDH (BBI Life Sciences, D110016-0100).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!