Proteins were extracted from NIH/3T3 cells and Raw264.7 cells using RIPA lysis buffer and quantified by the BCA protein assay kit. Western blots were performed using the standard sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis method and enhanced chemiluminescence detection reagents (Thermo, WP20005) (Zhou et al., 2019 (link)). Primary antibodies against the following proteins were used: rabbit anti-mouse p-PI3K antibody (Abcam, Cat. ab182651, 1:1000 dilution), rabbit anti-mouse PI3K antibody (Abcam, Cat. ab86714, 1:1000 dilution), rabbit anti-mouse p-Akt antibody (Abcam, Cat. ab222489, 1:1000 dilution), rabbit anti-mouse Akt antibody (Abcam, Cat. ab8805, 1:1000 dilution), rabbit anti-mouse β-actin (Sigma, Cat. A5441, 1:50,000 dilution). As a secondary agent, the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, 7074, dilution 1: 5,000) was used. Quantitative analyses were performed using ImageJ software, with β-actin as the internal standard.
Wp20005
Manufactured by Thermo Fisher Scientific
Sourced in United States
The WP20005 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for specific laboratory applications. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Ab8245, by Abcam (4 mentions)
Ea03752box, by Thermo Fisher Scientific (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Rabbit anti mouse β actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Pr1910, by Solarbio (1 mentions)
Ta 125 tt, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Ibright cl750, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
P2438, by Merck Group (1 mentions)
P0670, by Beyotime (1 mentions)
Rabbit anti β actin, by Merck Group (1 mentions)
Ab125066, by Abcam (1 mentions)
Ab62331, by Abcam (1 mentions)
Ab37185, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
19 protocols using wp20005
1
Western Blot Analysis of PI3K/Akt Signaling
2
Quantitative Immunoblotting of Melanoma Cells
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After transfection, A375 cells and A2058 cells were lysed with RIPA Buffer (89900, ThermoFisher), and total protein lysates were then quantitated by BCA kits (A53227, ThermoFisher). The protein lysates (45 μg) and marker (4 μl; PR1910, Solarbio) were loaded separately and subjected to electrophoresis with 10% SDS‐PAGE gel (P0670, Beyotime). Later, the protein was transferred onto PVDF membranes (P2438, Sigma‐Aldrich) and blocked by 5% skim milk with Tris‐Buffered Saline with 1% Tween 20 (TBST; TA‐125‐TT, ThermoFisher) for 1 h. Afterward, the membranes were incubated with primary antibodies for TUBB (#2128, 55 kDa, 1:1000, Cell Signaling Technology), and GAPDH (#4292, 37 kDa, 1:500, Cell Signaling Technology) overnight at 4°C. The membranes were again washed with TBST and added with Goat anti‐Rabbit IgG (A32731, 1:10,000, ThermoFisher). An enhanced chemiluminescence reagent kit (WP20005, ThermoFisher) was used to develop immunoblots, which were later analyzed on an imaging device (iBright CL750, ThermoFisher), and quantified by ImageJ software (1.52s version, National Institutes of Health).
3
Western Blotting for PI3K/Akt Signaling
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Western blotting was performed according to standard protocols (Tadokoro et al., 2020 (link)). The following antibodies were used: rabbit anti-PI3K antibody (Abcam, Cat. # ab86714, 1:1,000 dilution), rabbit anti-p-Akt antibody (Abcam, Cat. # ab222489, 1:1,000 dilution), rabbit anti-β-actin (Sigma, Cat. # A5441, 1:50,000 dilution). As secondary agents, horseradish peroxidase-coupled goat anti-rabbit antibody was used. Bands were visualized with an enhanced chemiluminescence detection kit (Thermo, WP20005). All results were quantified using ImageJ software, with β-actin as the internal standard.
4
Western Blot Analysis of Ferroptosis Markers
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HK-2 cells and RCC cells were homogenized in RIPA lysis buffer containing protease inhibitors. We measured the protein concentration of each sample by bicinchoninic acid (BCA) assay and then were separated by SDS-PAGE. Transferred onto a piece of polyvinylidene difluoride transfer membrane, proteins of each sample were blocked with 5% nonfat milk for 1 h. The membranes were incubated with antibodies against MITD1 (PA5-116854, Cell Signaling Technology), SLC7A11 (ab37185, Abcam), glutathione peroxidase 4 (GPX4; ab125066, Abcam), cyclooxygenase 2 (COX2; ab62331, Abcam), acyl-CoA synthetase long-chain family member 4 (ACSL4; ab155282, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab8245, Abcam). After incubation with primary antibodies for one night, the membranes were washed and incubated with secondary antibodies. The protein bands were visualized using enhanced chemiluminescence reagents (WP20005, Thermo Fisher Scientific). Finally, using ImageJ software performed the densitometric analysis to quantify differences in protein levels.
5
Western Blot Analysis of MSH3, SLC7A11, and GAPDH
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First, we homogenized HK-2 cells and RCC cells in RIPA lysis buffer containing protease inhibitors. Then, we measured the protein concentration of each sample using bicinchoninic acid (BCA) and then separated it using SDS-PAGE. The protein of each sample was transferred to a polyvinylidene fluoride transfer membrane and sealed with 5% skimmed milk for 1 hour. The membrane was incubated with antibodies to resist MSH3 (ab69619, Abcam, UK), SLC7A11 (ab175186, Abcam, UK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab8245, Abcam, UK). After incubating with the first antibody overnight, the membrane was washed and incubated with the second antibody. Protein bands were visualized using enhanced chemiluminescence reagents (WP20005, Thermo Fisher Scientific, USA).
6
Quantitative Western Blot Protocol
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Cellular protein samples are isolated from tissue and whole-cell lysates and quantified using the Pierce BCA Protein Assay Kit (23227, Thermo Fisher, USA). Tissue and cell proteins are extracted using RIPA buffer. Twenty micrograms of protein from each sample is loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk for 1 h, incubation is performed using the following antibodies: mouse anti-GAPDH (ab8245, Abcam, Cambridge, UK), Smad2/3 (ab202445, 1:100, Abcam, Cambridge, UK), LIF (sc-515931, 1:100, Santa Cruz Biotechnology Co., Ltd, China). All antibodies are diluted according to the instructions. On the following day, imaging is captured using an enhanced chemiluminescence reagent (WP20005, Thermo Fisher, USA) and a ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad Laboratories, California, Hercules, USA). The Western blot images are quantitatively analyzed for grayscale values of bands from each group using Image J analysis software, with GAPDH as an internal reference. The experiment is repeated three times. All Original western blots images can be found in the Supplementary files.
7
Western Blot Analysis of Cellular Proteins
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Samples of both tissues and cells were lysed in RIPA lysis buffer containing protease inhibitors. Proteins of different molecular weights were separated by SDS-PAGE. Transferred to a polyvinylidene fluoride (PVDF) membrane, the proteins of each sample were blocked with 5% skimmed milk for 1 h. The membranes were incubated with the primary antibodies targeting LAMA2 (ab236762, Abcam, UK), E-cadherin (ab76319, Abcam, UK), N-cadherin (ab76011, Abcam, UK), Vimentin (ab8069, Abcam, UK), and GAPDH (ab8245, Abcam, UK). After incubation with primary antibody overnight, the membrane was washed with TBST and incubated with secondary antibody. Protein bands were visualized using an enhanced chemiluminescence reagent (WP20005, Thermo Fisher Scientific, USA). Finally, densitometric analysis was performed using ImageJ software to quantify differences in protein levels.
8
Western Blot Analysis of Protein Expression
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The concentration of total cell protein was measured by BCA protein detection kit (Beyotime, China, Jiangsu). Then, the proteins were dispersed by size via 10% SDS-PAGE, and the resultant bands were transferred to PVDF membranes. After being blocked with 5% skimmed milk powder supplemented with TBST buffer, the membranes were incubated with primary antibodies at 4°C overnight. The main antibodies are mTOR, S6K1, p-mTOR (Ser 2448), p-S6K1 (Thr 389), β-actin (Cell signal Technology Company, Boston, USA), BDNF (Affinity, USA), and MBP (Proteintech, USA). The membranes were then incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody. The banding patterns were finally visualized with enhanced chemiluminescence (WP20005, Thermo Fisher Scientific, California, United States), and densitometry was analyzed by VisionWorks software (Eastman Kodak Company, New York, United States).
9
AAV Transduction Validation via VEGFR3 Serum Protein
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To validate AAV transduction, whole blood was collected from the thoracic cavity prior to cardiac perfusion, followed by coagulation (30 min, room temperature) and serum (supernatant) collection. Serum was purified by centrifugation (two times for 10 min, 2000 g , 4°C) and consecutive collection on ice. Bromophenol blue and 5% β-mercaptoethanol were added to the obtained serum, boiled at 95°C for 5 min and used to detect the soluble VEGFR31–4-Ig or VEGFR34–7-Ig serum proteins by western blotting. Recombinant mouse VEGFR3 chimera protein (R&D Systems, 743-R3-100) was used as quantitative standard. Proteins were separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes. Mouse VEGFR3 domains 1−4 and 4−7 were detected by probing with the polyclonal goat anti-mouse VEGFR3 antibody (R&D Systems, AF743, 1:1000) against the extracellular domain of VEGFR3. Signal detection was done using donkey anti-goat secondary antibodies conjugated to horseradish peroxidase (0.375 µg/mL, Jackson ImmunoResearch) in combination with enhanced chemiluminescence solution (ThermoFisher Scientific, WP20005). Visualization was performed on a ChemiDoc MP imaging system (Biorad).
10
Protein Extraction and Western Blot Analysis
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Clinical samples were lysed with RIPA lysis buffer including protease and phosphatase inhibitor for 10 min under ice. After centrifugation at 12,000×g for 10 min at 4°C, the supernatants were collected and quantitied based on BCA protein detection kit. Next, PVDF membranes were blocked in 5% bovine serum albumin (BSA) and incubated with LNPEP antibodies (BOSTER, A05092-1) at 4°C overnight. Subsequently, after incubation with HRP conjugated secondary antibody for 45min at room temperature, the image signals were detected with a chemiluminescence reagent (Thermofisher, WP20005).
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