Total protein was extracted using a protein extraction kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol, and the protein concentration was determined using an ultraviolet spectrophotometer (Onedrop1000; Shanghai Genechem Co., Ltd.). A total of 10 µg protein/lane was loaded on a 12% polyacrylamide gel, resolved using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Sangon Biotech Co., Ltd.). The PVDF membrane was blocked with 5% non-fat milk for 2 h at room temperature. Subsequently, the PVDF membrane was incubated with the corresponding primary antibody against the target protein overnight at 4°C followed by incubation with the secondary antibody at room temperature for 1 h. The protein band was detected using a Beyo ECL Star kit (Beyotime Institute of Biotechnology). The antibodies used were as follows: Anti-PI3K (ab70912; 1:100), anti-MEK1 (ab32091; 1:1,500), anti-ERK1 (ab32537; 1:600), anti-cdc25 (ab111830; 1:2,000) (all Abcam), anti-C-fos (554C1a; 1:500; Santa Cruz Biotechnology, Inc.), anti-ALOX12B (PA5-23608; 1:800; Invitrogen; Thermo Fisher Scientific, Inc.), anti-GAPDH (ab181602; 1:10,000; Abcam), goat anti-mouse IgG antibody (ab97035; 1:2,000) and goat anti-rabbit IgG antibody (ab7090; 1:5,000) (both Abcam).
Ab32091
Manufactured by Abcam
Sourced in United States
Ab32091 is a laboratory equipment product. It is a versatile tool designed for use in scientific research and experiments. The core function of this product is to [core function]. Further details about its intended use or applications are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab96379, by Abcam (5 mentions)
Ab184699, by Abcam (3 mentions)
Ab8227, by Abcam (2 mentions)
Ab111830, by Abcam (1 mentions)
Protein extraction kit, by Yeasen (1 mentions)
Ab70912, by Abcam (1 mentions)
Ab97035, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Pvdf membrane, by Sangon (1 mentions)
Ab7090, by Abcam (1 mentions)
Ab32537, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Ab124957, by Abcam (1 mentions)
Ab38515, by Abcam (1 mentions)
Ab32041, by Abcam (1 mentions)
Ab214036, by Abcam (1 mentions)
Ab36988, by Abcam (1 mentions)
Ab59195, by Abcam (1 mentions)
Ab65854, by Abcam (1 mentions)
Ab175134, by Abcam (1 mentions)
9 protocols using ab32091
1
Western Blot Analysis of Protein Targets
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After lysing in RIPA buffer, the collected total protein was separated on 12% SDS-PAGE and shifted onto PVDF membranes. 5% nonfat milk powder was added for blocking membranes. Next, the membrane was probed all night at 4 °C with primary antibodies against loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA), TRAP (ab65854, 1/1000; Abcam), NFATc1 (ab175134, 1/1000; Abcam), TRAF3 (ab36988, 1/1000; Abcam), TRAF1 (ab129279, 1/1000; Abcam), TRADD (ab110644, 1/1000; Abcam), TRAF2 (ab230795, 1/1000; Abcam), ADAM17 (ab39162, 1/1000; Abcam), TNFRII (ab8161, 1/1000; Abcam), TNFRSF1A (ab19139, 1/1000; Abcam), p-IKB (ab133462, 1/1000; Abcam), IKB (ab32518, 1/1000; Abcam), TNFRSF11 (NB100-56508, 1/1000; Novus Biologicals, Littleton, CO, USA), p-IKKα (ab38515, 1/1000; Abcam), IKKα (ab32041, 1/1000; Abcam), p-IKKβ (ab59195, 1/1000; Abcam), IKKβ (ab124957, 1/1000; Abcam), p-Erk (ab214036, 1/1000; Abcam), Erk (ab184699, 1/10000; Abcam), p-MEK (ab96379, 1/1000; Abcam), MEK (ab32091, 1/1000; Abcam). On the following day, the HRP-secondary antibodies were added for 2 h at room temperature. The protein density was examined by the ECL luminous liquid (Pierce, Rockford, IL, USA). The assay was taken in triplicate.
3
Skin Tissue Protein Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Skin tissues were homogenized with CelLyticTM MT Cell Lysis Reagent (Sigma-Aldrich). Ten micrograms of the protein lysate was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies against PCNA (1:1,000; ab29, Abcam), Cyp1a1 (1:1,000; #871, Daiichi Pure Chemical Co., Ltd., Tokyo, Japan), Cyp1b1 (1:1,000; ab185954, Abcam, Cambridge, UK), Actin (1:1,000; sc1616-HRP, Santa Cruz Biotechnology, Santa Cruz, CA), Pan-Ras (1:1000; Component of STA-440, Cell Biolabs, Inc., San Diego, CA), EGF receptor (1:1000; #2232, Cell Signaling Technology, Danvers, MA), phosphorylated (Y1086) EGF receptor (1:1,000; ab32086, Abcam), MEK1 (1:1,000; ab32091, Abcam), phosphorylated (E342) MEK1 (1:1,000; ab396379, Abcam), Cleaved Caspase-3 (1:5000; #9661, Cell Signaling Technology), Flag-M2 (1:1000; F3165, Sigma-Aldrich), and p16INK4a (1:500; sc1207, Santa Cruz Biotechnology). Specific antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies and Chemi-Lumi One (Nacalai Tesque) or ImmunoStar reagent (Wako, Osaka, Japan).
4
Immunoblotting of Key Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins from lymphocytes were isolated by RIPA (Biorbyt, orb402072) protein lysate, separated by 10% SDS-PAGE gel electrophoresis, and transferred onto a membrane for 90 min. Then, the membrane was blocked at room temperature for 1 h using 1% BSA (Solarbio, PC0001), primary antibody dilutions MEK (Abcam, ab32091), ERK1/2 (Abcam, ab17942), p-MEK (Abcam, ab96379), p-ERK1 (Abcam, ab200807), p-ERK2 (Abcam, ab151549), PI3K (Abcam, ab106693), Akt (Abcam, ab2732), mTOR (Abcam, ab182651), p-Akt (Abcam, ab192623), p-mTOR (Abcam, ab137133), and β-actin (Abcam, ab8227) were subsequently added and incubated at room temperature for 2 h. After washing the membrane three times with TBST buffer, the cells were added with HRP-labeled secondary antibody (Abcam, ab205718) and incubated at room temperature for 0.5 h, then washed with TBST buffer for three times. The protein bands were developed by ECL luminescence kit (Absin, abs920) for detection.
5
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
By reported procedures,18 (link) protein extraction and Western blotting were performed. The subfamily of pERK is ERK1 (pT202/pY204)+ Erk2 (pT185/pY187) (cat. no ab5001, dilution, 1:10,000; Abcam, Cambridge, MA, USA), the subfamily of ERK is ERK1+ ERK2 (cat. no ab115799, dilution 1:1,000; Abcam). The subfamily of pMEK is MEK1 (pS298) (cat. no ab96379, dilution 1:3,500; Abcam), and the subfamily of pMEK is MEK1 (cat. no ab32091, dilution 1:5,000; Abcam). β-Actin was considered as the internal control. All results were conducted in triplicate.
6
Immunohistochemical Analysis of PA28γ, MEK1, and Ki67
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining of PA28γ, MEK1, p-MEK1, and Ki67 was performed following the protocols of previous reports. Paraffin-embedded sample sections were cut at 4 μm thickness, deparaffinized by heating at 60–65 °C (approximately 30 min), deparaffinized in xylene, and hydrated. Heat-mediated antigen retrieval was performed (5 min), and sections were immersed in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. The slides were incubated at 4 °C overnight with primary antibody diluted with 3% BSA solution. The primary antibody information is as follows: PA28γ (PA5-21789, Invitrogen, Carlsbad, USA), 1:200; MEK1 (ab32091, Abcam, Cambridge, UK), 1:200; p-MEK1 (ab96379, Abcam, Cambridge, UK), 1:200; and Ki67 (ab66155, Abcam, Cambridge, UK), 1:100. Then, tissue sections were incubated with the matching secondary antibodies (Zsbio, Beijing, CHN) for 30 min at 37 °C and visualized with 2,3-diaminobenzidine (DAB) hydrochloride. Images of the tissue sections were acquired with an Aperio Scanscope (Aperio, USA). IHC scores were based on the staining intensity and density of positive cells, which were assessed and confirmed by two experienced pathologists independently.
7
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected with 10 nM siPOOL in 6 well plates at 6 × 104 cells/well density and incubated at 37°C with 5% CO2 for 72 h. Cells were lysed in a buffer containing 10 mM Tris–HCl, 1 mM EDTA, and 1% SDS. Protein concentration was determined with the BCATM protein assay kit (Thermo Fisher, 23227). Home cast 10% SDS gels or Novex 4–20% 10 well Mini Gels (Thermo Fisher, XP04200) were used for SDS–PAGE. Transfer was done using PVDF membranes and a Trans‐Blot SD Semi‐Dry Electrophoretic Transfer Cell (Bio‐Rad). Imaging was done with an Odyssey Fluorescence scanner (Li‐COR) (Figs 4C and EV2B ). The following primary antibodies were used: anti‐total ERK (M7927, Sigma), anti‐MEK1 (ab32091, Abcam), anti‐MEK2 (ab32517, Abcam), anti‐BRAF (sc‐5284, Santa Cruz), anti‐CRAF (9422S, Cell Signaling Technology), anti‐SOS1 (610096, Biosciences), anti‐GRB2 (PA5‐17692, Invitrogen), and anti‐RSK2 (sc‐9986, Santa Cruz). Anti‐GAPDH (sc‐32233, Santa Cruz) or anti‐Actin (A2066, Merck) was used as protein of reference. For the secondary antibodies, we used the IRDye 680LT donkey anti‐mouse IgG (926‐68022, Li‐COR), IRDye 800CW goat anti‐mouse (926‐32210, Li‐COR), and IRDye 800CW donkey anti‐rabbit (926‐32213, Li‐COR). Protein quantification was done with the Image StudioTM Lite software.
8
Comprehensive Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ice-cold RIPA buffer (Cell Signaling, USA) was used to lyse the cells. Total protein lysates (50 μg) were separated by denaturing 10% SDS-PAGE and electrotransfered onto a nitrocellulose membrane (Millipore, USA) for subsequent blotting with primary antibodies. The primary antibodies were: IMP4 (1 : 1000, ab181046; Abcam), Bcl-2 (1 : 1000, #15071; CST), Bax (1 : 1000, ab32503; Abcam), PARP (1 : 1000, ab191217; Abcam), E-cadherin (1 : 1000, 20874-1-AP; Proteintech, Wuhan), N-cadherin (1 : 1000, ab280375; Abcam), Vimentin (1 : 1000, ab20346; Abcam), glucose transporter 1 (GLUT1; 1 : 1000, ab115730; Abcam), hexokinase II (HK2; 1 : 1000, ab209847; Abcam), cleaved fructose phosphate kinase P (PFKP; 1 : 1000, ab32561, Abcam), pyruvate kinase M2 (PKM2; 1 : 1000, ab85555; Abcam), lactate dehydrogenase A (LDHA; 1 : 1000, #3582; Cell Signaling, USA), MEK1 (1 : 1000, ab32091; Abcam), p-MEK1 (1 : 1000, ab96379; Abcam), ERK (1 : 1000, ab184699; Abcam), p-ERK (1 : 1000, ab201015; Abcam), p21 (1 : 1000, ab109520; Abcam), p53 (1 : 1000, ab75754; Abcam), cyclin D1 (1 : 1000, ab16663; Abcam), and GAPDH (1 : 1000, ab181603; Abcam), according to the manufacturers' protocols. After incubation with secondary antibodies for 60 min, protein bands were visualised using ECL kit (Beyotime).
9
Hippocampus and Striatum Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins in the hippocampus and striatum cells were extracted. Primary antibodies CB1 (ab23703, abcam), MEK1 (ab32091, abcam), p-MEK1 (ab214445, abcam), ERK1/2 (ab184699, abcam), p-ERK1/2 (ab32538, abcam) and β-actin (ab8227, abcam) were used. The Western blot procedure is described above.
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!