Total protein lysates were prepared from lung tissues of ALI mice using RIPA Buffer (89900, ThermoFisher, USA) and quantitated using a BCA protein assay kit (A53227, ThermoFisher, USA). Forty microliter of protein and 5 μl of marker (PR1910, Solarbio, China) were separately loaded on 12% SDS-PAGE gel (P0053A, Beyotime, Shanghai, China) and subjected to electrophoresis. Afterwards, the protein was transferred onto PVDF membranes (P2438, Sigma-Aldrich, USA), and the membranes were blocked by Tris Buffered Saline and 1% Tween 20 (TBST, TA-125-TT, ThermoFisher, USA) containing 5% skimmed milk. Then, the membranes were incubated at 4℃ overnight with primary antibodies against Occludin (SAB35 00301, 57 kDa, 1：1,000, Sigma-Aldrich, USA), SPC (ab2 11326, 21 kDa, 1：1,000, Abcam, Cambridge, MA, USA), FGF2 (SAB2108135, 31 kDa, 1：1,000, Sigma-Aldrich, USA), and GAPDH (ab181602, 36 kDa, 1：10,000, Abcam, USA). Then, the membranes were incubated with Goat Anti-Rabbit IgG (A32733, 1：20,000, ThermoFisher, USA) at room temperature for 1 hour. The immunoreactive bands were detected on an imaging system (iBright CL1500, A44240, ThermoFisher, USA) using an enhanced chemiluminescence reagent kit (WP20005, ThermoFisher, USA). The density of the bands was quantified by ImageJ software (version 1.52s, National Institutes of Health, Bethesda, MD, USA).
Pr1910
Manufactured by Solarbio
Sourced in China
The PR1910 is a laboratory centrifuge designed for general-purpose applications. It features a compact design and can accommodate a variety of sample tubes and microplates. The centrifuge provides controlled speed and time settings to enable efficient sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Ta 125 tt, by Thermo Fisher Scientific (3 mentions)
P2438, by Merck Group (3 mentions)
Ab181602, by Abcam (2 mentions)
Bca kit, by Thermo Fisher Scientific (2 mentions)
P0670, by Beyotime (2 mentions)
Ipfl00010, by Merck Group (2 mentions)
E cadherin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ibright cl1500, by Thermo Fisher Scientific (1 mentions)
Ab211326, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ibright cl750, by Thermo Fisher Scientific (1 mentions)
Wp20005, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab7090, by Abcam (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab52903, by Abcam (1 mentions)
8 protocols using pr1910
1
Western Blot Analysis of Lung Proteins
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 Blot Analysis of Apoptosis and Signaling Pathways
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The total protein of cells and rat kidney tissues was extracted with RIPA Lysis Buffer (C500007, Sangon, China), and the protein concentration was determined using BCA assay kit (C503021, Sangon, China). An equivalent of 30 μg protein extract and 5 µL marker (PR1910, Solarbio, China) were resolved on the SDS-PAGE, followed by being transferred onto PVDF membrane (IPFL00010, Millipore, USA). Next, the protein blot was blocked with skim milk, and probed with primary antibodies, followed by further incubation with appropriate secondary antibody goat anti-rabbit IgG (1:2,000, ab7090; abcam, UK). Subsequently, immunoreactive bands were detected using an ECL kit (ab133409; abcam). GAPDH served as an internal control. The protein bands on X-ray films were quantified with a Tanon 5200 imaging system (Tanon, China). The primary antibodies from abcam and Cell Signaling Technology (CST) are listed as follows: Bax (1:2,000; Rabbit; ab182733, 21 kDa; abcam), Bcl-2 (1:500; Rabbit; ab194583, 26 kDa; abcam), Cleaved Caspase 3 (1:1,000; Rabbit; #9661, 17 kDa; CST), p-Smad3 (1:2,000; Rabbit; ab52903, 48 kDa; abcam), Smad3 (1:1,000; Rabbit; ab40854, 48 kDa; abcam), p-protein kinase A (p-PKA, 1:1,000; Rabbit; #5661, 42 kDa; CST), PKA (1:1,000; Rabbit; #4782, 42 kDa; CST), NADPH oxidase 4 (Nox4, 1:1,000; Rabbit; ab154244, 67 kDa; abcam), and GAPDH (1:10,000; Rabbit; ab181602, 36 kDa).
4
Protein Profiling of Soymilk Enzymes
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The effect of enzyme treatments on protein profiles of soymilk and enzyme modified soymilk was determined using SDS-PAGE with separating and stacking gels containing 15 and 4% acrylamide, respectively. A molecular weight marker ranging from 11 to 180 kDa (PR1910, Solarbio, Beijing, China) was used as a standard. Skim milk, soymilk and enzyme modified soymilk were dissolved with sample buffer (10 mM DTT, pH 6.8, 1 mM EDTA, 1% SDS, 10% glycerol, and 0.01% bromphenol blue) and then boiled for 5 min. After centrifugation, the electrophoresis was carried out following the method of Lamsal et al. [37 (link)].
5
Western Blot Protein Analysis Protocol
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For the western blot [21 (link)], after the cells were fully lysed, total protein was extracted using RIPA lysate (P0013, Beyotime, CA). Then the protein loading buffer was added to the protein and mixed, and then the mixture was boiled for 10 min. Next, a 15 μg protein sample was added to each well and electrophoresed (at 80 V for 40 min, at 120 V for 80 min), and susbsequently transferred to a PVDF membrane (at 210 mA for 100 min) (Immobilon-P Transfer Membrane, EMD Millipore Corporation, MA). The membrane was blocked with 8% skim milk for 2 h, and then incubated with the primary antibody overnight at 4 °C, followed by incubation with the corresponding secondary antibody for 2 h. An ECL kit (#6883, Cell Signaling Technology, USA) was used for chemiluminescence development, and ImageJ (version 5.0, Bio-Rad, USA) software was used for semi-quantitative analysis. The primary antibodies were as follows: E-Cadherin (1:10,000, 97kD, ab40772, Abcam, UK), N-Cadherin (1 µg/ml, 130kD, ab18203), Vimentin (1:1000, 54kD, ab92547), GAPDH (1:500, ab8245, 36kD), cleaved Caspase-3 (1 µg/ml, 17kD, ab2302), Bcl-2 (1:500, 26kD, ab59348), Bax (1:1000, 21kD, ab32503); the secondary antibodies were Goat Anti-Mouse IgG H&L (1:5000, ab205719) and Goat Anti-Rabbit IgG H&L (1:5000, ab205718); and the markers were PR1910 (11-180kD) and PR1920 (11-245kD) (Solarbio, CA).
6
Western Blot Analysis of GLP1R and PKA Signaling
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The procedures of western blot were performed as described previously.18 Briefly, the EC cells were lysed and total protein was extracted by Radio‐Immunoprecipitation Assay (RIPA) lysis buffer (P0013K, Beyotime). After quantification of protein concentration, equal amounts of marker (5 μl; PR1910, Solarbio) and protein (45 μg) were fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (IPFL00010, Millipore). Subsequently, the membranes were blocked and cultured with primary antibodies at 4°C overnight, including those against GLP1R (1:500; Rabbit; bs‐1559R, 51 kDa, Bioss), phosphorylated‐PKA (p‐PKA; 1:1000; Rabbit; ab32390, 45 kDa, Abcam), PKA (1:10000; Rabbit; ab32514, 45 kDa, Abcam), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 1:1000; Mouse; ab8245, 37 kDa). After being washed by tris‐buffered saline tween (TBST) buffer, the membranes were incubated with goat anti‐rabbit (1:2000, ab6721, Abcam) and rabbit anti‐mouse (1:3000, ab6728, Abcam) secondary antibodies for 2 h. The relative intensity of the protein bands was quantified using Tanon 5200 Imaging System (Tanon), with GAPDH serving as the loading control.
7
Protein Expression Analysis in C666-1 and HK1 Cells
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Processed C666‐1 cells and HK1 cells were lysed by RIPA Buffer (89,900, ThermoFisher) blended with a Protease‐Phosphatase inhibitor (A32959, ThermoFisher). The concentration of total protein lysate was determined using the BCA kit (A53227, ThermoFisher). The total protein (45 μg) and marker (5 μl) (PR1910, Solarbio) were separately loaded, separated by 10% SDS‐PAGE gel (P0670, Beyotime), and transferred onto PVDF membranes (P2438, Sigma‐Aldrich). Then, the membranes were blocked by 5% skim milk in Tris Buffered Saline with 1% Tween 20 (TBST, TA‐125‐TT, ThermoFisher) at room temperature for 1 h. Afterwards, the membranes were incubated at 4°C overnight with primary antibodies against AKT (rabbit, ab8805, 55 kDa, 1:500, Abcam), phosphorylated (p)‐AKT (rabbit, ab38449, 56 kDa, 1:500, Abcam), PI3K (rabbit, #4292, 85 kDa, 1:1000, Cell signaling technology), p‐PI3K (rabbit, #4228, 85 kDa, 1:1000, Abcam), and GAPDH (mouse, ab8245, 36 kDa, 1:10000, Abcam). After TBST washing, the membranes were incubated with secondary antibody Goat antiRabbit IgG (A32731, 1:10000, ThermoFisher) or Goat antiMouse IgG (A32733, 1:1000, ThermoFisher). The protein bands were visualized using the enhanced chemiluminescence reagent (WP20005, ThermoFisher) and analyzed using ImageJ software (1.52 s version, National Institutes of Health). All protein expressions were normalized to GAPDH.
8
Quantitative Protein Expression Analysis
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Total protein was extracted with RIPA reagent (P0013B, Beyotime Biotechnology, China), and the protein concentration was measured using a BCA protein detection kit (P0012, Beyotime Biotechnology, China). Equal amounts of total protein 30 µg were then loaded with size markers (PR1910, Solarbio, China), and 10% SDS-PAGE was performed. Then, proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk for 1 hour at room temperature and washed with TBST for 5 minutes. The blocked membrane was incubated overnight at 4 • C with primary antibody (FOXM1, 1:2000; Abcam, MA, USA; E-cadherin, 1:3000; Abcam; N -cadherin, 1:4000; Abcam; vimentin, 1:4000; Abcam; Fibronectin, 1:5000; Abcam; GAPDH, 1:4000, Abcam) and washed four times (5 min each), followed by in-cubation with secondary antibody (1:5000, Abcam) at room temperature for 1 hour. After further TBST washes, protein bands were detected using an enhanced chemiluminescence kit (WBKLS0500, MILLIPORE, USA) and enhanced Chemiluminescence Advanced System (Bio-Rad, CA, USA) and quantified with Image J software (National Institutes of Health, MD, USA). In addition, GAPDH served as an internal control for quantifying the target proteins.
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