RNA extraction and q-PCR were performed as described previously25 . Expression of STIM1 and HIF-1α was detected using a TaqMan q-PCR assay system (Applied Biosystems, Foster City, CA, USA). Western blot was performed as previous described25 . The antibodies used in this study were: STIM1 (Cell Signaling Technology, 5668), HIF-1α (Abcam, 113642), E-cadherin (Cell Signaling Technology, 3195), and vimentin (Cell Signaling Technology, 49636).
Stim1
Manufactured by Cell Signaling Technology
Sourced in United States
STIM1 is a calcium sensor protein that plays a crucial role in the regulation of cellular calcium homeostasis. It functions by detecting changes in intracellular calcium levels and initiating a signaling cascade that activates calcium-dependent processes within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Stim2, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (3 mentions)
Orai1, by Merck Group (3 mentions)
Grp78, by Cell Signaling Technology (2 mentions)
Serca2, by Cell Signaling Technology (2 mentions)
Bcl xl, by Cell Signaling Technology (2 mentions)
Orai1, by GeneTex (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Taqman qpcr assay system, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Hif 1α, by Abcam (1 mentions)
Flag hrp, by Merck Group (1 mentions)
Ha hrp, by Roche (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
20 protocols using stim1
1
Analyzing STIM1 and HIF-1α expression
2
Comprehensive Protein Detection Techniques
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For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, ♯sc-47778; 1:5000), ATF6 (Cell Signaling Technology, ♯65880; 1:1000), BCL-2 (AbCam, ♯ab182858; 1:500), BCL-XL (Cell Signaling Technology, ♯2764; 1:1000), Cleaved-Caspase-8 (Cell Signaling Technology, ♯8592; 1:1000), Caspase-3 (Cell Signaling Technology, ♯9665; 1:1000), Caspase-9 (Cell Signaling Technology, ♯9508; 1:1000), CHOP (Cell Signaling Technology, ♯2895; 1:250), GFP (AbCam, ♯ab13970; 1:2000), GRP78 (Cell Signaling Technology, ♯3177; 1:1000), FLAG-HRP (Sigma Aldrich, ♯A8592; 1:4000), HA-HRP (Roche, ♯11867423001; 1:2000), IP3R1 (Thermo Fischer, ♯PA1-901; 1:1000), IP3R1 & IP3R2 [previously described77 ; 1:1000], IP3R3 (BD Biosciences, ♯610312; 1:1000), KDEL (AbCam, ♯ab12223; 1:2000), MCL-1 (Cell Signaling Technology, ♯5453; 1:1000), Nicastrin (BD Biosciences, ♯612290; 1:1000), PARP (Cell Signaling Technology, ♯9542; 1:1000), SERCA2 (Cell Signaling Technology, ♯4388; 1:1000), STIM1 (Cell Signaling Technology, ♯5668; 1:1000).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP3R1 (Thermo Fischer, ♯PA1-901; 1:200), IP3R3 (BD Biosciences, ♯610312; 1:200).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP3R1 (Thermo Fischer, ♯PA1-901; 1:200), IP3R3 (BD Biosciences, ♯610312; 1:200).
3
Western Blot Analysis of Cell Signaling Proteins
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For total protein analysis, cells were harvested with lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.4% β-Mercaptoethanol, Proteinase-Inhibitor Cocktail, Roche, Mannheim, Germany). Clarified protein lysate was applied to a polyacrylamide gel and analyzed by western blotting [95 (link), 96 (link)]. To this end, 30-50 μg protein of whole cell lysate was incubated with primary antibody for Orai1 (1:1000, Millipore, Bedford, MA, USA, [22 (link)]), STIM1 (1:1000, cell signaling, Danvers, MA, USA, [97 (link)]), Akt (1:1000, cell signaling, Danvers, MA, USA, [93 (link)], Phospho-Akt (Thr308) (1:1000, cell signaling, Danvers, MA, USA, [93 (link)]and GAPDH (1:1000, cell signaling, Danvers, MA, USA, [98 (link)]). For detection secondary antibody conjugated with horseradish peroxidase (HRP) (1:2000, Cell Signaling, Danvers, MA, USA) was used. Antibody binding was identified with ECL detection reagent (Amersham, Freiburg, Germany). Bands were quantified with Quantity One Software (Biorad, München, Germany [22 (link)]). The appropriate band has been defined by using Orai1 overexpressing cells [22 (link), 24 (link)].
4
Western Blot Analysis of RNA-Binding Proteins
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Cells were lysed in IP lysis buffer as described Srikanth, 2010 #420}. Whole cell lysates were separated on 4–12% Bis-Tris gels (Life Technologies) and proteins were transferred to PVDF membranes (Biorad). Blocking was done using superblock (5% low fat milk, 2% Casein and 1% BSA) or 5% low fat milk Membranes were incubated with the primary antibody in 3% BSA (w/v) in TBST (137 mM NaCl, 20 mM tris, 0.1% Tween-20, pH 7.6) overnight at 4 °C. Membranes were incubated with the HRP-conjugated secondary antibody for one hour at room temperature and bound proteins were visualized using ECL Western blotting detection reagents (GE Healthcare). Blots were imaged using Geliance Gel Imaging system (Perkin Elmer).
Primary antibodies used were STIM1 (4916 S Cell Signaling), Ago2 (2897 S Cell Signaling), β-actin (A1978 from Sigma), Dicer (5362 S Cell Signaling), Drosha (3364 S Cell Signaling), Pumilio1 (12322 S Cell Signaling) and Pumilio2 (12323 S Cell Signaling) Secondary antibodies used were from Jackson ImmunoResearch Laboratories goat anti-rabbit IgG (111-035-044) and goat anti-mouse IgG (115-035-146).
Primary antibodies used were STIM1 (4916 S Cell Signaling), Ago2 (2897 S Cell Signaling), β-actin (A1978 from Sigma), Dicer (5362 S Cell Signaling), Drosha (3364 S Cell Signaling), Pumilio1 (12322 S Cell Signaling) and Pumilio2 (12323 S Cell Signaling) Secondary antibodies used were from Jackson ImmunoResearch Laboratories goat anti-rabbit IgG (111-035-044) and goat anti-mouse IgG (115-035-146).
5
Western Blot Analysis of Ion Channels
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Samples of PV tissue or PVSMCs were homogenized or lysed in buffer containing 62.5 mM Tris·HCl (pH 6.8), 2% SDS, 10% glycerol, 5% protease inhibitor cocktail, 1 mM EDTA, and 200 M 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Protein concentration was measured by using the BCA Protein Assay Kit (Bio-Rad) with BSA as a standard (Calbiochem, San Diego, CA). The homogenized proteins were mixed with 150 mM dithiothreitol, heated at 95°C for 3 minutes, and resolved by 10% SDS-PAGE. These separated proteins were then transferred to polyvinylidene difluoride membranes (pore size 0.45 µm, BioRad) and incubated with rabbit polyclonal antibodies against TRPC1 (Sigma), TRPC6 (Sigma), STIM1 (Cell Signaling) or Orai1 (Alomone labs) for overnight or mouse monoclonal antibody that was specific for α-actin (Sigma) for 1 hour. Secondary antibodies were probed with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (KPL, Gaithersburg, MD) for 1 hour and then detected using an enhanced chemical luminescence system (GE Healthcare, Piscat away, NJ).
6
Oxaliplatin and CTSS Inhibitor Synthesis
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Oxaliplatin was purchased from Sanofi Pharmaceutical Company (NY, USA). The procedure for synthesizing the CTSS inhibitor RJW-58 was described previously 14 (link). The following primary antibodies were purchased: Cathepsin S (Thermo Fisher catalog: #PA5-81369), EBF (Santa Cruz, #sc-137065), ATF-3 (Biossua, #BS-0519R), α-tubulin (Sigma, MO, USA), IRF-1 (Bio-Rad, #VPA00801), CBP (GeneTex, #GTX101249), Iba1 (Abcam, #ab-5076), NeuN (Novus, #NBP2-10491), IL-10 (Abcam, #ab189392), STIM1 (Cell signaling (D88E10), #5668S), CD11b (Abcam, #ab52478), CD45 (BD Biosciences, #553079), STAT3 (BD, #50402), P-STAT3 (Cell signaling, #9145S), NFκB (Cell signaling, #4727S), OR5B3 (Abcam, #ab186624), OR5M3 (LSBio, #LS-C200406), and β-actin (GeneTex, #GTX109639). Horseradish-peroxidase-conjugated secondary antibodies were purchased from GeneTex (#GTX213110, #GTX213111, #GTX224125, and #GTX232040).
7
Protein Extraction and Signaling Pathway Analysis
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Proteins were extracted using 1X RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA), and concentration determined using the BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA). Immunoblots (western blot) were performed as previously described [48 (link)]. Primary antibodies against ERK1/2, p-ERK1/2 (Thr202/Tyr204), MEK1/2, p-MEK1/2 (Ser217/221), p-PKCα/β II (Thr638/641), CAMKII, p-CAMKII (Thr286), BIM; p-BIM (Ser69), CHOP, Bcl-2, Bcl-xL, p-eIF2α (Ser51), STIM1, PARP, NEDD8 and secondary HRP-conjugated antibodies were obtained from Cell Signaling (Danvers, MA). Antibodies for PKCβ, luciferase, ERO1 and Orai1 were purchased from Santa Cruz Biotechnology (Dallas, TX). Proteins expression was determined by densitometry analysis of the immune-detected bands, normalized to β-actin, and presented relative to control (fold induction). Co-IP assays were carried out using the Pierce™ Classic Magnetic IP Kit (Thermo Fisher Scientific Inc., Waltham, MA) as described [49 (link)].
8
Immunostaining Protocol for STIM1, STIM2, and β-Tubulin III
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The modified AbCam protocol for immunostaining (www.abcam.com , accessed on 22 July 2019) was used. Cells on coverslips were fixed with 4% formaldehyde for 10 min, permeabilized with 0.25% TritonX-100 for 10 min, blocked with 10% FBS and 0.3M glycine in PBST (PBS with 0.1% Tween20) for 1 h, and subsequently incubated with antibodies in 1% BSA in PBST for 1 h. Primary antigen-specific antibodies included: STIM1 (Cell Signaling №5668, 1:800), STIM2 (Cell Signaling №4917, 1:50), and neuron-specific β-tubulin III (Sigma T8578, 1:500). Secondary antibodies were Alexa Fluor 555 anti-rabbit (Thermo Fisher Scientific, Waltham, USA, A-21428, 1:500) and Alexa Fluor 488 anti-mouse (Thermo Fisher Scientific A-11001, 1:2000). Nuclei were counterstained with DAPI in the mounting medium. Images were obtained with an IX83 microscope and Olympus FV3000 confocal laser system using an Olympus 40x objective (oil, 1.3 NA); the pinhole was one Airy disk. Fluorescence was excited by 405, 488, and 561 nm laser lines and detected. Representative z-stack images were captured using Olympus FluoView software (v2.1.1.98) and analyzed with FiJi software (v1.53f51) [72 (link)].
9
Autophagy Regulation by AMPK and SOCE
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Reagents: AICA-riboside (#2627-69-2), ATRA (#CR2625); adensine 5’-triphosphate (ATP) disodium salt hydrate (#34369-0708); BAPTA-AM [1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl) ester] (#126150-97-8); Benzonase endonuclease (#71205); 8-CPT-cAMP, 8-(4-chlorophenylthio) 3’,5’-cyclic adenosine monophosphate (#C3912); metformin (#PHR1084); and thapsigargin (#67526-95-8) were purchased from Sigma-Aldrich/Merck Millipore, France. STO-609 (#S8274) was purchased from Selleckchem, USA; BTP2, N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl] phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (#3939) and 2-APB (2-aminoethoxydiphenyl borate) (#1224) were purchased from TOCRIS Bioscience, France. Tetra-methylrhodamine methyl ester (TMRM) (#T-668) and propidium iodide (# P3566) were purchased from Life Technologies, France. Antibodies against the following proteins were used: phospho-AMPK, Thr 172 (#2531), AMPK (#2532), phospho-CAMKK2 (Ser511) (#12818), and LC3A/B (#4108) and STIM1 (#5668) were purchased from Cell Signaling Technology, USA; CAMKK2 (#ab168818) was purchased from Abcam, UK; LC3 (#L7543) and ORAI1 (# 08264) were purchased from Sigma-Aldrich, France; p62/SQSTM1 (#610832), CD11c (#333145) and CD11c (#560895) were purchased from BD biosciences, France. Cell culture media were purchased from Life Technologies-Invitrogen, France.
10
Immunoblotting for Signal Transduction
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Protein samples (60 μg) were heated to 95 °C for 5 min and loaded onto 10% sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% nonfat dry milk for 1 h at room temperature. Membranes were washed with 0.1% PBST (phosphate-buffered saline (PBS) and Tween20) three times and then incubated with primary antibodies overnight at 4 °C. Antibodies against pERK1/2 (Cell Signaling, Beverly, MA), STIM1 (Cell Signaling, Beverly, MA), and ORAI1 (GeneTex, Hsinchu, Taiwan) were diluted 1: 2000, whereas the antibody against β-actin was diluted 1: 10000. Membranes were then washed with 0.1% PBST three times and incubated with a 1: 5000 dilution of anti-mouse or anti-rabbit HRP-conjugated immunoglobulin G (IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. After washing with 0.1% PBST three times, signals were detected by an ECL-plus Western blotting detection system (Millipore).
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