Proteins were extracted by collecting cells in RIPA buffer (Sigma-Aldrich) containing Complete Protease Inhibitor Cocktail (Roche), and protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad). Proteins were detected using the following antibodies: KRAS (sc-30) from Santa Cruz.; VEGF-A (ab46154, sc-7269) from Abcam and Santa Cruz; and β-actin (A5441) from Sigma.
β actin a5441
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, China
β-actin (A5441) is a cytoskeletal protein that is ubiquitously expressed in eukaryotic cells. It is commonly used as a reference or control protein in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
α tubulin t5168, by Merck Group (3 mentions)
Cleaved caspase 3 9661, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Vinculin v9131, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Phospho akt1, by Cell Signaling Technology (2 mentions)
Vdac 4866, by Cell Signaling Technology (2 mentions)
α tubulin t9026, by Merck Group (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Hydroxyurea, by Merck Group (2 mentions)
α tubulin, by Merck Group (2 mentions)
Lamp1, by Abcam (2 mentions)
Lps derived from e coli 0111 b4 l2630, by Merck Group (2 mentions)
Luria bertani broth l8050 luria bertani agar, by Teknova (2 mentions)
88 protocols using β actin a5441
1
Cell Protein Extraction and Antibody Detection
2
Quantifying Phospho-Akt Levels in 3T3-L1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates of 3T3-L1 cells were collected. Protein levels were determined by blotting with phospho-Akt (S4060, Ser473) were purchased from Cell Signaling Tschnologies. β-actin (A5441) were purchased from Sigma–Aldrich. Hmgcs2 (ab137043) were purchased from Abcam. Chemiluminescent images were obtained by ChemiDoc Touch imaging system (Biorad, Hercules, CA) with Optimal Auto-exposure mode.
3
Western Blot Analysis of PI3K/Akt Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10–20 µg of total protein was separated with 7.5–12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% non-fat milk (LabScientific, Highlands, New Jersey) in TBS for 30 min and then incubated with primary antibodies in TBS containing 5% milk and 0.1% Tween-20 overnight at 4°C. After rinsing with TBS-0.1% Tween, membranes were incubated with the secondary antibodies diluted in 5% milk-TBS for 1.5 hr at RT. Bands were detected by using Odyssey CLx Scanner. Band intensity quantifications were performed using Image Studio 3.1. Normalizations were performed either relative to the actin or Akt band intensities. Western blots were performed with antibodies against p110α (#4249), p110β (#3011), Akt (#4691), phospho-Akt T308 (#13038), phospho-Akt S473 (#9271), phospho-Erk 1/2 T202/Y204 (#9101), phospho-S6 S235/236 (#2211), phospho-S6 S240/244 (#5364), phospho-EGFR Y1068 (#2236), S6 (#2217), HA (#2367), TfnR (transferrin receptor) (#13208), Nup (nucleoporin) (#2598), EGFR (#4267), PTEN (#9188) (all Cell Signaling), Rac1 (05–389) (Millipore, Billerica, Massachusetts), β-actin (A5441), α-tubulin (T5168) (Sigma), Caveolin1 (MA3-600) (Thermo Fisher) and Gq (sc-392) (Santa Cruz). IRDye 800CW Goat anti-mouse or anti-rabbit secondary antibodies (Li-Cor, Lincoln, Nebraska) were used.
4
Inflammasome Activation by LPS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LPS from Escherichia coli 0111:B4, the antibody against β-actin (A5441), SP600125, PD98059, and cholesterol-water soluble were obtained from Sigma-Aldrich. Antibodies against phospho-p38 MAPK (Thr180/Tyr182; 4511), p38 MAPK (9212), phospho-p44/42 MAPK (Thr202/Tyr204; 9101), p44/42 MAPK (4695), phospho-SAPK/JNK (Thr183/Tyr185; 4668), SAPK/JNK (9252), and IL-1β (12242) were purchased from Cell Signaling Technology. Antibodies against ASC (22514), and active caspase-1 (sc-514 and 515) were obtained from Santa Cruz Biotechnology, Inc. Specific ON-Target plus siRNAs against murine mRNAs were obtained from Thermo Fisher Scientific. Silencer Select siRNAs specific to decrease the expression of human LPIN2 mRNA and negative controls were purchased from Ambion. Nigericine, the specific caspase-1 inhibitor Ac-YVAD-AOM, and SB203582 were purchased from EMD Millipore, and monosodium urate and alum were obtained from InvivoGen. The general caspase inhibitor Z-VAD-FMK was from APExBIO. The rest of the reagents were purchased from Sigma-Aldrich.
5
Western Blotting Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues or cells were lysed and then denatured at 100 °C for 10 min. The protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore Corp), and immunoblotted with the following primary antibodies: SENP2 (YT4237) was from Immunoway; phospho-AMPKα (2535S), AMPKα (5832S), phosphor-Raptor (2083), Raptor (2280), and HA tag (3724) were from Cell Signaling Technology; G6PC (A16234) and PCK1 (A2036) were from Abclonal; FLAG tag (F1804) and β-actin (A5441) were from Sigma–Aldrich; Myc tag (E022050-02) was from EΛRTH, and PCx (16588-1-AP) was from Proteintech. The specificity and reproducibility of the used antibodies were validated.
6
Multimodal Mitochondrial Function Analysis
7
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blot analyses, cells were lysed in lysis buffer (137 mM NaCl, 10% glycerol, 20 mM Tris–HCl pH 8.0, 2 mM EDTA pH 8.0, 1% Igepal, 5 µL protease inhibitor cocktail [Sigma-Aldrich]) for 20 min on ice. After centrifugation (15 min at 17,000g, 4°C) the protein content of the samples was determined in three technical replicates according to the Bradford method. 10–20 µg protein were loaded onto precast gels and blotted onto PVDF membranes (both Bio-Rad). Primary antibodies targeting hnRNP DL (sc-133699, Santa Cruz), hnRNP D (07-260 Merck Millipore), GFP (118144600, Roche), β-actin (A-5441, Sigma-Aldrich), or HSP60 (ab6530, Abcam) were used. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch) were used as secondary antibodies. Blots were developed with the ECL system (Bio-Rad) or Amersham ECL select (GE Healthcare) for weaker signals. Images were detected using the ChemiDoc Imaging System (Bio-Rad).
8
Mitochondrial Dynamics Regulatory Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in the present studies included polyclonal antibodies to optic atrophy 1(OPA1) (80471S), LC3 (12741S), p62 (5114S), DRP1 (8570S), voltage‐dependent anion channel (VDAC) (4866S), cleaved caspase‐3 (9661S), Bax (2772S), Bcl‐2 (2876S), and PINK1 (6946S), all of which were purchased from Cell Signaling (Danvers, MA, USA). Mitofusin‐2 (MFN2) (Ab56889), Parkin (Ab15954), α‐synuclein (Ab1903), and phosphor S129 α‐synuclein (Ab51253) were purchased from Abcam (Cambridge, UK). TH (T1299), NeuN (ABN90), and β‐actin (A5441) antibodies were purchased from Sigma‐Aldrich. CCK8(C0038) was purchased from Beyotime Biotechnology (Shanghai, China). GFAP (SC‐71143) and Tom 20 (SC‐136211) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A320 (ALCAT1 inhibitor) was obtained from Perenna Pharmaceuticals Inc (San Antonio, TX, USA). ALCAT1 antibody was obtained from Dr. Arai (University of Tokyo, Japan).
9
Protein Expression Analysis in EndoC-βH2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EndoC-βH2 cells were lysed in RIPA buffer (Sigma-Aldrich). Protein concentrations were quantified by Pierce BCA protein assay kit (Thermo Fischer Scientific). Proteins were resolved by SDS-PAGE and transferred to a membrane using the iBlot2 Gel transfer device (Thermo Fischer Scientific). Western blots were performed using the following primary antibodies diluted (1/1000) in TBS 3% BSA 0.1% Tween-20: phospho-rpS6 (Ser240/244: D68F8), total rpS6 (5G10), 4EBP1 (53H11), and p44/42 MAPK (Erk1/2) (9102S) (all from Cell Signaling); anti-puromycin (MABE343-12D10) and anti-Prohormone convertase PCSK1 (AB10553) (both from Merck Millipore); anti-PRKAR2A (612243) from (BD Biosciences). α-TUBULIN (T9026) and β-ACTIN (A5441) (both from Sigma-Aldrich) were diluted (1/2000) in TBS 5% milk 0.1% Tween-20. After washing, membranes were incubated with species-specific horseradish peroxidase–linked secondary antibodies (Cell Signaling) diluted (1/2000–1/10,000) in TBS 1% milk 0.1% Tween-20 and visualized on an ImageQuant LAS 4000 following ECL exposure (GE Healthcare). Densitometric quantification of Western blots was done using ImageJ software and normalized to α-tubulin expression. Immunoblotting experiments were performed three times.
10
Cell Cycle Regulation Assay Protocol
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!