Dulbecco’s Modified Eagle’s Medium (DMEM; high glucose), trypsin/EDTA solution, and fetal bovine serum (FBS) were purchased from Gibco® (Thermo Fisher Scientific, Waltham, MA, USA). Cell Counting Kit-8 (CCK)-8 and the Annexin V Apoptosis Detection kit I were acquired from MultiSciences Biotech Co., Ltd. (Hangzhou, Zhejiang, People’s Republic of China). ALO, PCNA, Bax, and Bcl-2 antibodies were purchased from Abcam, Inc. (Cambridge, UK). Caspase-3, Caspase-9, Ras, p-Raf1, Raf1, Erk1/2, p-Erk1/2, and GAPDH antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). These antibodies were monoclonal and derived from rabbit. Hoechst 33,342 kits were obtained from Wuhan Boster Biological Technology Ltd. (Wuhan, China). The EZ-Cytox cell viability assay kit (MTT) was acquired from MultiSciences Biotech Co., Ltd. (Hangzhou, China). Trans-well chambers were obtained from Corning Co. (Corning, NY, USA). The protein assay kit was obtained from Bio-Rad Labs. (Hercules, CA, USA).
P raf1
Manufactured by Cell Signaling Technology
Sourced in United States
P-Raf1 is a laboratory equipment product used for the detection and quantification of phosphorylated Raf-1 protein. It is designed to provide researchers with a reliable tool for investigating cell signaling pathways involving the Raf-1 kinase and its phosphorylation status.
Automatically generated - may contain errors
Lab products found in correlation
Raf 1, by Cell Signaling Technology (3 mentions)
P erk1 2, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Erk1 2, by Cell Signaling Technology (3 mentions)
Mmp 9, by Cell Signaling Technology (2 mentions)
P mek, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Transwell chamber, by Corning (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Cell counting kit 8 cck 8, by MultiSciences Biotech (1 mentions)
Rac1 cdc42, by Cell Signaling Technology (1 mentions)
P mek1 s298, by Cell Signaling Technology (1 mentions)
Ipa 3, by Selleck Chemicals (1 mentions)
8 protocols using p raf1
1
Assessing Apoptosis and Proliferation in Cancer Cells
2
Molecular Pathways in Cell Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IPA-3 and U0126 were purchased from Selleck Chemicals (Shanghai, China). Antibodies and their sources are as follows: antibodies against p-PAK1 (T423, #2601), PAK1 (#2602), PAK2 (#4825), PAK3 (#2609), Rac1/Cdc42 (#4651), Raf1 (#9422), p-Raf1 (S338, #9427), MEK1 (#2352), p-MEK1 (S298, #9128), ERK1/2 (#4695), p-ERK1/2 (T202/Y204, #4370), MMP-2 (#4022) and MMP-9 (#3852) were obtained from Cell Signaling Technology (Beverly, MA). Primary antibodies against p-PAK2 (S141, #SAB4504634) and Actin (#4700) were purchased from Sigma-Aldrich (Shanghai, China).
3
RNA Expression and Protein Analysis of Th Cell and DC Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qPCR was performed as previously described10 (link). Total RNA was extracted from cells by using an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. The mRNA levels of Dectin1, Il9, Ifng, Il4, Il5, Il13, Il17, Tnfsf15, Ox40l, Spi1, Irf4, Tbx21, Gata3 and Rorc by Th cells or DCs were analysed. Expression was normalized to the expression of the house-keeping gene Gapdh. Primer sets used for these analyses are listed in Supplementary Table 1 .
Western blot assay was performed as previously described10 (link). Anti-mouse phosphorylated (p)-Syk, Syk, pRaf1, Raf1, p-IKKα/β, IκB-α, p65, p50, c-Rel, RelB, p52, β-actin and HDAC1 antibodies were purchased from Cell Signaling Technology (CST). RIPA Buffer (cat #: 9806) were purchased from CST. Nuclear extraction kit (cat #: 78833) was purchased from Thermo Scientific. Images have been cropped for presentation. Full-size images are presented inSupplementary Figs 14 and 15 .
Western blot assay was performed as previously described10 (link). Anti-mouse phosphorylated (p)-Syk, Syk, pRaf1, Raf1, p-IKKα/β, IκB-α, p65, p50, c-Rel, RelB, p52, β-actin and HDAC1 antibodies were purchased from Cell Signaling Technology (CST). RIPA Buffer (cat #: 9806) were purchased from CST. Nuclear extraction kit (cat #: 78833) was purchased from Thermo Scientific. Images have been cropped for presentation. Full-size images are presented in
4
Western Blot Primary and Secondary Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used for western blotting include Flag M2 at 1:3000 (Sigma, F3165, St. Louis, MO), Flag-HRP at 1:1000 (Sigma, A8592, St. Louis, MO), GST Z-5 at 1:3000 (Santa Cruz, sc-459, Dallas, TX), rabbit GST-HRP at 1:1000 (Sigma, A7340, St. Louis, MO), Aurora kinase A at 1:500 (Cell Signaling, 4718, Boston, MA), rabbit pERK and ERK (Cell Signaling, 4370, 9102, respectively, Boston, MA), pMEK and MEK (Cell Signaling, 9154, 4694, respectively, Boston, MA), pRaf-1 (Cell Signaling, 9427, Boston, MA) and Raf-1 (Santa Cruz, sc-133, Dallas, TX) at 1:1000, and Ras (BD Transduction Laboratories, 610002, San Jose, CA) at 1:1000. Secondary antibodies include goat anti-rabbit IgG (Santa Cruz, sc-2004, Dallas, TX) and goat anti-mouse IgG (Santa Cruz, sc-2005, Dallas, TX) and were used at either 1:2500 or 1:5000 dilutions. Antibodies used for immunoprecipitation include Aurora A (Sigma, A1231, St. Louis, MO), Ras (ThermoScientific, MA1-012X, Waltham, MA), and IgG (Santa Cruz, sc-2027, Dallas, TX) at 1:50 dilution.
5
Immunoblotting Analysis of HIV Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblotting (IB) analyses under reducing (Fig. 2a ) or nonreducing (Fig. 2b ) conditions, DCs (2 × 106 cells/well) were cultured in starvation medium (RPMI–0.5% FCS) for 3 h and thereafter infected with HIV or HIV-C (500 ng/ml p24) or left untreated and uninfected (iDC). For MAVS, 9 h after infection, cells were lysed and proteins analyzed. IB was done as described by Posch et al. (2 (link)). For IP, whole-cell extracts from 2 × 106 HIV-1 infected DCs were used, and IP was performed with 5 μg anti-MAVS (sc-166583; Santa Cruz) used to coat protein G agarose beads (11719394001; Roche). Abs used for IB and IP were phospho-Thr592 SAMHD1 Ab (kindly provided by F. Diaz-Griffero) and SAMHD1, pSAPK/JNK, pERK1/2, pPLK1, pRaf-1, MAVS, pIRF3, NF-κB, TBK1, ERK1/2, and tubulin Abs (all from Cell Signaling Technology). The RIG-I Ab MA5-31715 was from Invitrogen. For every donor, quantification was performed using ImageJ and a loading control to normalize the values (ERK1/2 and tubulin). Quantifications were performed using values from at least 3 donors.
6
Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis and cell sample harvesting and preparation were performed by a standard procedure as presented previously [28 (link)]. We used primary antibodies against HIF-1α (GeneTex, USA), HIF-2α, Nanog (Novus Biologicals, USA), β-actin, Oct4 (Santa Cruz), p-Akt (S473), Akt, p-STAT3 (Y705), STAT3, p-ERK (T202/Y204), ERK, p-glycogen synthase kinase 3 (S9) (p-GSK3β), RAF1, p-RAF1 (S259), p-RAF1 (S338), early growth response protein 1 (EGR1), DUSP6, p-MAP/ERK (S217/221) (p-MEK), and MEK (all Cell Signaling Technology, USA). Following immunodetection, each membrane was stained by Amido black to confirm the transfer of the protein samples. The total level of β-actin was detected as loading control. Typical representative western blots from at least three independent experiments are shown. Densitometry analysis was performed using ImageJ software (NIH, USA) and relative protein expression was calculated after normalization to the total protein of interest or β-actin. Data are presented as the mean + SEM.
7
Antibody-based Signal Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies that recognize ERK1/2, p-ERK1/2, RAF1, p-RAF1, AKT, p-AKT, MEK1/2, p-MEK1/2, GAPDH, COXIV, VDAC1, cleaved caspase-3, calnexin, PCNA, E-cadherin and vimentin were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against β-actin, Myc, and Flag were obtained from Sigma-Aldrich (St Louis, MO). Antibodies that recognize cyclin D1 and CDK4 were purchased from Santa Cruz Biotechnology (Dallas, TX). ATAD3A antibody was purchased from Novus Biologicals (Abingdon, UK). CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was obtained from Promega (Madison, WI). Texas-red phalloidin, D-luciferin bioluminescent substrate, and alamarBlue Cell Viability Reagent were purchased from ThermoFisher Scientific (Waltham, MA). The RAS inhibitor salirasib and the ERK1/2 inhibitor SCH772984 were obtained from SelleckChem (Houston, TX), and 4-nitroquinoline-1-oxide (4NQO) was purchased from Sigma-Aldrich (St. Louis, MO).
8
Studying DMDD's Influence on 4T1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Different groups of cells were given the corresponding concentrations of DMDD and cultured for 24 hours, and then different groups of 4T1 cells were separately digested and collected into centrifuge tubes. Highly efficient RIPA tissue/cell rapid lysate (Solarbio, Beijing, China) was mixed with PMSF (Solarbio, Beijing, China) and a protease inhibitor cocktail (CWBIO, Beijing, China) at a ratio of 100:1:1 and the mixture were used to extract proteins from 4T1 cells. BCA Protein Assay kit (Beyotime, Shanghai, China) was applied to detect the protein concentration. Protein samples (50 µg) were electrophoresed on SDS-PAGE gels (10%), and protein signal was transferred onto PVDF membranes (ISEQ00010; Millipore, Billerica, MA, USA) by electroblotting. The membranes were soaked in primary antibodies at 4°C overnight and then soaked in secondary antibody for 1 hour at room temperature on the shaker. Primary antibodies included p-raf1, raf1, p-mek, mek, perk, erk, p-JNK, JNK, p-p38, p38, Bcl2, Bax, MMP2, MMP9 and GAPDH (all from Cell Signaling Technology, MA, USA). The ratio of all primary antibody to diluent is 1:1000. Secondary antibody was anti-rabbit (5366P) IgG(H+L) (DyLight(TM)) antibodies (1:10,000). Reactive bands of the membranes were detected using a near-infrared two-color fluorescence imaging system (LI-COR Odyssey CLx). GADPH was used as an internal reference.
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!