Pericytes were incubated for 48 h with 50 ng/ml FGF-2 in DMEM containing 2% FBS prior to stimulation with 10 ng/ml PDGF-BB for 10 min. For time-course experiments, cells were treated for 10 min, 1 and 6 h with 50 ng/ml FGF-2, 50 ng/ml PDGF-BB, or a mixture of FGF-2 and PDGF-BB. Non-treated pericytes were used as a control. Soluble proteins from total cell lysates were applied to an SDS-PAGE (NP0301; Invitrogen), followed by wet transferring onto a methanol-activated polyvinylidene fluoride membrane (LC2002, Invitrogen). Membranes were blocked at room temperature with 3% skim milk for 60 min, followed by incubation overnight with a rabbit anti-mouse PDGFRβ (1:1000; 28E1; Cell Signaling) antibody, a rabbit anti-mouse phospho-PDGFRβ antibody (1:1000; G63G6; Cell Signaling), or an anti-mouse β-actin antibody (1:1000; 3700S; Cell Signaling). Membranes were incubated at room temperature for 60 min with a mixture of secondary antibodies consisting of an anti-mouse secondary antibody conjugated with IRDye 680RD (1:15,000; LI-COR; Lincoln) and an anti-rabbit secondary antibody conjugated with IRDye 800CW (1:15,000; LI-COR; Lincoln). Protein signals were captured using an Odyssey CLx system (LI-COR). Full-gel images are shown in Supplementary Fig. S6 .
Lc2002
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LC2002 is a liquid chromatography system designed for analytical and preparative chromatography applications. It features a compact and modular design, providing reliable performance and ease of use.
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Lab products found in correlation
Anti rabbit igg antibody, by Cell Signaling Technology (2 mentions)
Imagequant ecl imager, by GE Healthcare (2 mentions)
Ab8245, by Abcam (2 mentions)
Signalfire ecl reagent, by Cell Signaling Technology (2 mentions)
Odyssey clx system, by LI COR (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Ecl chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Ab190242, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Fnn0021, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
11 protocols using lc2002
1
Pericyte response to FGF-2 and PDGF-BB
2
ASFV Georgia 2007/1 Protein Detection
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Lysates from ASFV Georgia 2007/1 (VERO cell-adapted)-infected VERO cells were used to perform a western blot as previously described [11 ]. Briefly, the prepared cell lysates were electrophoresed on a NuPAGE 4–12% Bis-Tris Gel (1.0mm X 2D well) for 35 mins, followed by transfer to 0.2 μm PVDF membranes (Invitrogen #LC2002) for 1 hour. The membranes were then blocked for 1 hr. in blocking buffer (PBST+5% non-fat dry milk) and transferred to the Protean II Slot-Blotter. Sera from week 2 post-boost were diluted 1:250 in blocking buffer and added to individual wells for 1 hr. at room temperature with shaking. After washing the wells 3X with PBST, the membranes were removed from the blotting apparatus and incubated for 1 hr. with a 1: 2,000 dilution of Goat anti-swine-HRP (KPL #14-14-06). Following washes, the membranes were developed using DAB (Sigma #D4293). ASFV-specific convalescent serum (1:10,000) was used as a positive control and normal swine serum (1:200) was used as a negative control. Background reactivity to host-cell antigens was gauged similarly using mock-infected lysates. The western blot analysis was carried out at Plum Island Animal Disease Center.
3
Western Blot Analysis of FAT-1 Protein
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The tissues were homogenized by bead milling for approximately 5 min at 4°C, and the cells were treated with lysis buffer for 30 min on ice. Proteins were then collected by centrifugation at 1,000 × g, at 4°C for 30 min. Subsequently, proteins (approximately 50 µg) were denatured, separated on 12% SDS/PAGE gels, and then transfected onto a PVDF membrane (LC2002, Invitrogen; Thermo Fisher Scientific, Inc.). This was followed by blocking of the membranes with 5% fat-free milk. Primary antibodies to FAT-1 (1:2,000, ab190242, Abcam) and GAPDH (1:2,000, ab8245, Abcam) were then separately incubated with the membrane for >8 h at 4°C. Subsequently, anti-rabbit IgG antibody (1:5,000, 7074, Cell Signaling Technology, Inc.) and anti-mouse IgG antibody (31430, Thermo Fisher Scientific, Inc.) were incubated with the membrane at room temperature for 2 h. Finally, the protein signal of the membrane was detected using ECL Chemiluminescent Substrate (WP20005, Thermo Fisher Scientific, Inc.) and analyzed using an ImageQuant ECL Imager (28-9605-63, GE Healthcare).
4
Western Blot Analysis of Cell Protein Markers
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The cells were gently washed by 1× PBS twice and treated by 1% NP-40 (FNN0021, Invitrogen, ThermoFisher, USA) supplemented with halt protease inhibitor cocktail (78429, Thermo Scientific, ThermoFisher, USA) on ice and proteins were collected by centrifugation at 1,000 g, 4 °C for 30 min. The concentration of the proteins was measured by Pierce™ BCA Protein Assay Kit (23225, Thermo Scientific, ThermoFisher, USA). Next, 25 µg protein extraction were separated on 10% SDS-PAGE gel and then transferred to PVDF membranes (LC2002, Invitrogen, Thermofisher, USA). The membranes were washed by 1xTBST (50 mM Tris, 150 mM NaCl and 2% Tween-20; pH 7.5) for three times at room temperature and then incubated with primary antibodies (Anti-Ki-67 (ab15580, 1:2,000, Abcam, UK), Anti-PCNA (ab92552, 1:2,000, Abcam, UK) and Anti-GAPDH (ab181602, 1:5,000, Abcam, UK)) at 4 °C overnight. Next, the membranes were further incubated with anti-rabbit IgG antibody (1:2,000, 7074, Cell Signaling Technology, USA) at 4 °C overnight and with SignalFire™ ECL reagent (6883, Cell Signaling Technology, USA) for 1min at room temperature in the dark, and then exposed to X-ray. Images were captured by ImageQuant ECL Imager (28-9605-63, GE Healthcare, USA) and analyzed by using Image J software v.1.48. GAPDH was used for normalization.
5
Protein Expression Analysis Protocol
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Total proteins were extracted from the tissues or cells using RIPA buffer (89901, Thermo Scientific, USA) containing protease inhibitors (36978, Thermo Scientific, USA). The supernatant was then collected by centrifugation at 4°C for 30 min at 12,000 × g. The concentration of the protein was measured using the BCA Protein Assay Kit (P0012S, Beyotime). The proteins (50 μg per sample) were isolated on 10–12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (LC2002, Invitrogen, ThermoFisher, USA), which was blocked by 5% non-fat milk (PA201-01, BioMed, China) at room temperature for 1 h. The following primary antibodies were used for subsequent incubation: anti-PTGS2 antibody (ab15191, Abcam, UK); anti-HMGB1 antibody (ab18256, Abcam, UK); anti-GPX4 antibody (ab125066, Abcam, UK); anti-FTH1 antibody (ab183781, Abcam, UK) and anti-GAPDH antibody (ab8245, Abcam, UK). After incubation with the primary antibodies at 4°C overnight, the membrane was washed and probed by appropriate HRP-conjugated antirabbit IgG antibody (1:5,000, 7,074, Cell Signaling Technology, USA) for PTGS2, GPX4, and FTH1 or HRP-conjugated antiMouse IgG antibody (1:5,000, 70-GAM007, MultiSciences, China) at room temperature for 2 h. GAPDH served as an internal control. SignalFire™ ECL reagent (6,883, Cell Signaling Technology, USA) was used to detect the signals.
6
Aspirin Modulates BMAL1 Acetylation
7
Western Blot Analysis of Protein Expression
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As previous study, RIPA lysis buffer was used to extract total protein from mouse BMDMs and heart tissues [25 (link)]. Protein concentration was detected using a BCA kit. Cell or tissue lysates were separated using 10% SDS-PAGE. After the protein transfer step, PVDF membranes (LC2002, ThermoFisher) were blocked with 5% skimmed milk and then membranes were probed with primary antibodies against IDO (86,630, CST, USA), Bax (ab32503, Abcam, UK) or Bcl2 (ab196495, Abcam, UK). β-Actin served as a loading as control. After three washes in PBS buffer, secondary antibodies were incubated for 1 hour at room temperature. Lastly, proteins were visualized using ECL solution (WBKLS0100, Beijing Xinjingke Biotechnologies Co., Ltd., China). Blots were analyzed using Image J 3.0 (IBM, USA).
8
Western Blot Analysis of Proteins
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All proteins from cells or tumors were resolved by SDS-PAGE gel (P1200, Solarbio, Beijing, China) and transferred onto PVDF membranes (LC2002, Thermo, Waltham, US). Then, the membranes were blocked in 5% fat-free milk in TBST for 1 hour at room temperature and then washed, and primary antibodies were incubated at 4°C overnight. After being washed, the PVDF was incubated with horseradish peroxidase-conjugated secondary antibodies. The bands were visualized by ECL reagent (32106, Thermo). Antibodies are as follows: KIF11 (1 : 1000 dilution, ab61199, Abcam, Cambridge, UK); anti-β-actin (1 : 1000 dilution, ab5694, Abcam, Cambridge, UK).
9
Western Blot Analysis of Cleaved Caspase-3
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Transfected HEK 293 cells were lysed in 4× sample loading buffer and separated by SDS-gel electrophoresis on a 12.5% polyacrylamide gel and electroblotted to polyvinylidene difluoride membranes (Thermo Fisher Scientific, LC2002). Similar loading and transfer of proteins were confirmed by staining the membranes with Amido Black. After a 1 h blocking step in 5% milk powder in Tris-buffered saline with 0.05% Tween-20, membranes were incubated overnight with a rabbit polyclonal antibody to cleaved caspase-3 (Asp175) diluted 1:1000 (Cell Signaling Technology, 9661S). After incubation for 1 h with anti-rabbit IgG coupled to horseradish peroxidase (MP Biomedicals, Burlingame, USA, 0855689), blots were developed using SuperSignal (Thermo Fisher Scientific, 34080) followed by exposure to Kodak BioMax MR Films (Sigma-Aldrich, Z350397).
10
Western Blot Analysis of Histone Modifications
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Adherent cells were offline activated as described above, then washed in ice-cold PBS. Cells were then lysed in RIPA buffer (Thermo Fisher 89900) with benzonase nuclease (Sigma Aldrich E1014-5KU) and scrape harvested to generate whole-cell lysates. Samples were heated to 95 °C for 5 min before running on a 4–12% Bis-Tris gradient gel (Thermo Fisher NP0322) and transferring to a PVDF membrane (Thermo Scientific LC2002). Blots were then blocked in 5% bovine serum albumin (Sigma Aldrich A9418) in TBST (TBS with 0.1% Tween). The blots were then incubated with primary antibody overnight at 4 °C with gentle rocking (H3 Cell Signal 4499, H2AK119Ub Cell Signal 8240, H3K27me3 Cell Signal 9733: 1:1000). The next day, the blots were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch 111-035-144: 1:2000) for 30 min. The blots were then washed with TBST and incubated with ECL (Thermo Fisher 32109) before imaging.
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