The cells were lysed in RIPA buffer (Beyotime, Haimen, China) containing 1% protease inhibitor PMSF (Beyotime) and centrifuged at 12,000 rpm for 10 min. The supernatant containing total proteins was aspirated and the protein concentration was determined. The total proteins were separated by SDS-PAGE (Beyotime) and then transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with primary antibodies against ROS1 (1:500, Sangon Biotech, Shanghai, China), cleaved-caspase-3 (1:1000, Abcam, Cambridge Science Park, Cambridge, UK), Bcl-2 (1:400, BOSTER, Wuhan, China), Bax (1:400, BOSTER), cleaved-PARP (1:1000, Abcam), E-cadherin (1:400, BOSTER), Vimentin (1:500, BIOSS, Beijing, China), N-cadherin (1:400, BOSTER), p-PI3K (1:500, BIOSS), PI3K (1:400, BOSTER), p-Akt (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA) and Akt (1:200, Santa Cruz Biotechnology) at 4°C overnight. After washing with TBS-Tween 20 buffer, the membranes were incubated with goat anti-rabbit IgG-HRP (Beyotime) at 37°C for 45 mins. The bands were developed using ECL solution (Beyotime).
P pi3k
Manufactured by Bioss Antibodies
Sourced in China, United States
P-PI3K is a recombinant protein that corresponds to the p110 catalytic subunit of phosphoinositide 3-kinase (PI3K). PI3K is an enzyme that plays a key role in cellular signaling pathways involved in cell growth, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Bioss Antibodies (3 mentions)
β actin, by Bioss Antibodies (3 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Gapdh, by Bioss Antibodies (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bcl2 associated x (bax), by Boster Bio (1 mentions)
Cleaved parp, by Abcam (1 mentions)
E cadherin, by Boster Bio (1 mentions)
N cadherin, by Boster Bio (1 mentions)
Bcl 2, by Boster Bio (1 mentions)
Vimentin, by Bioss Antibodies (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Ecl solution, by Beyotime (1 mentions)
Sds page, by Beyotime (1 mentions)
Goat anti rabbit igg hrp, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
P akt, by Affinity Biosciences (1 mentions)
12 protocols using p pi3k
1
Western Blot Analysis of ROS1 and EMT Markers
2
Western Blot Analysis of Rat Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rat hippocampal tissues were washed 2–3 times with pre-chilled PBS, cut into small pieces and homogenized by adding ten times the volume of tissue in RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Then the supernatant was collected by centrifugation and homogenization (12,000 rpm, 4°C, 10 min), which is the total protein of rat hippocampal tissue. The BCA protein assay kit (AS1086, ASPEN, Wuhan, China) was used for the quantification of total proteins. Samples were separated by SDS PAGE and electro-transferred onto PVDF (0.45 um) membranes (Servicebio,G6015-0.45, Wuhan, China) after activation with methanol. The membranes were then incubated overnight at 4°C with specific primary antibodies:CB1 (Servicebio,GB111214), PI3K(BIOSS,BSM-33219M), P-PI3K (BIOSS,bs5570R), AKT (Servicebio,GB11689), P-AKT (Affinity,AF0908), STAT3 (Servicebio,GB11176), P-STAT3 (RuiyingBiological, RLP0250), Bcl2 (Servicebio, GB113375), BAX (Servicebio, GB11690),ACTIN(Servicebio, GB12001). The membranes were washed three times with TBST for 10 min each. After washing, the membranes were incubated with HRP anti-conjugated secondary antibody (Servicebio, GB23303) for 1 h. The membranes were washed in the same manner as described above. Membranes were scanned using the Fluor Chem FC3 system (Protein Simple, United States).
3
Gastric Cancer Drug Resistance Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human gastric cancer cell line BGC823 was purchased from KeyGEN BioTECH Co., Ltd. (China).
The drug-resistant cell line BGC823/5-Fu was established by the low-dose multiple shock method in our previous research [17 (link)] and stored at -196 °C. The drug resistance index of BGC823/5-Fu was 13.
The serum-free medium (SFM) was composed of 95% DMEM/F12 (Gibco, USA), 1% 2 µg/ml EGF (Peprotech, USA), 1% 2 µg/ml bFGF (Peprotech, USA), 2% B27 (Gibco, USA), 1% 100 U/ml penicillin/streptomycin (Gibco, USA) and 0.4 U insulin (Sigma, USA). The IGF-1 was purchased from Sigma (USA). The P-gp, MRP1, PI3K, p-PI3K, AKT, p-AKT, Nrf2 and β-actin antibodies were purchased from Bioss, Inc. (China) and Proteintech Group, Inc. (USA). HRP-labeled Goat Anti-Rabbit IgG, HRP-labeled Goat Anti-Mouse IgG and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG were purchased from Beyotime Biotechnology (China). The Annexin V-FITC/PI Kit was purchased from KeyGEN BioTECH Co., Ltd. (China). CD44 MicroBead Kit was purchased from Miltenyi Biontec. (Germany). Lipofectamine2000 was purchased from Invitrogen (USA).
The drug-resistant cell line BGC823/5-Fu was established by the low-dose multiple shock method in our previous research [17 (link)] and stored at -196 °C. The drug resistance index of BGC823/5-Fu was 13.
The serum-free medium (SFM) was composed of 95% DMEM/F12 (Gibco, USA), 1% 2 µg/ml EGF (Peprotech, USA), 1% 2 µg/ml bFGF (Peprotech, USA), 2% B27 (Gibco, USA), 1% 100 U/ml penicillin/streptomycin (Gibco, USA) and 0.4 U insulin (Sigma, USA). The IGF-1 was purchased from Sigma (USA). The P-gp, MRP1, PI3K, p-PI3K, AKT, p-AKT, Nrf2 and β-actin antibodies were purchased from Bioss, Inc. (China) and Proteintech Group, Inc. (USA). HRP-labeled Goat Anti-Rabbit IgG, HRP-labeled Goat Anti-Mouse IgG and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG were purchased from Beyotime Biotechnology (China). The Annexin V-FITC/PI Kit was purchased from KeyGEN BioTECH Co., Ltd. (China). CD44 MicroBead Kit was purchased from Miltenyi Biontec. (Germany). Lipofectamine2000 was purchased from Invitrogen (USA).
4
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissue samples were collected and lysed in RIPA buffer containing proteinase inhibitors (Sigma, USA) and phosphatase inhibitors (CWBio, China). The protein extracts were separated on SDS-PAGE gels, followed by electrotransfering onto polyvinylidene fluoride membranes (Sigma, USA). After blocking with 5% fat-free milk for 2 h at room temperature, the membranes were incubated with primary antibodies and subsequently incubated with secondary antibody (Thermo Scientific, MA, USA). Finally bands were scanned with Bio-Rad Imager and individual band intensity was quantified with software of ImageJ. Antibodies were used as follows: caspase 3 (CST, USA), cleaved caspase 3 (CST, USA), GSDMC (Abclonal, China), GSDMD (Abclonal, China), GSDME (Abclonal, China), PI3K (Bioss, China), p-PI3K (Bioss, China), Akt (Proteintech, China), p-Akt (CST, USA), Bcl-2 (Proteintech, China), Bax (Proteintech, China) and β-Actin (Abclonal, China).
5
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% BSA for 1 h, then incubated with primary antibodies overnight at 4 °C. Antibodies were used as following: ERCC6L (Proteintech, 15,688–1-AP, 1:1000), PI3K (Bioss, bs-0128R, 1:1000), p-PI3K (Ser1070; Bioss, bs-6417R, 1:1000),AKT1 (Bioss, bs-0115R, 1:1000), p-AKT1 (Thr34; Bioss, bs-5194R, 1:1000), JAK2 (Bioss, bs-23003R, 1:1000), p-JAK2 (Tyr1007 + Tyr1008; Bioss, 2485R, 1:1000), NF-κB (Bioss, bs-0465R, 1:1000), p-NK-κB (Thr505; Bioss, bs-5663R, 1:1000), and β-actin (Bioss, bs-0061R, 1:5000). The next day membranes were incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C. After washed for 3 times in TBST for 5 min, membranes were visualized by chemiluminescence kit and scanned with QuantityOne software (Bio-Rad, Hercules, CA, USA). The bands were analyzed with ImageJ (NIH, Bethesda, MA, USA).
6
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted, and the concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States). Sample lysates (10 μg of protein) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with specific antibodies against MARCKS (1:2000) (Proteintech Group, China), p-MARCKS (1:1000) (Proteintech Group, China), PI3K (1:2000) (CST, United States), p-PI3K (1:500) (BIOSS, China), Akt (1:2000) (CST, United States), p-Akt (1:1000) (CST, United States), β-Catenin (1:2000) (CST, United States), p-β-Catenin (1:500) (CST, United States), Vimentin (1:2000) (CST, United States), matrix metalloproteinase (MMP)-9 (1:500) (Abcam, United States), N-Cadherin (1:1000) (Abcam, United States) or E-Cadherin (1:2000) (CST, United States) at 4 °C overnight, followed by incubation with the appropriate secondary antibody. Protein levels were normalized to those of total GAPDH, which was detected with a monoclonal anti-GAPDH antibody (1:10000) (Sigma-Aldrich Corporation, St. Louis, MO, United States). Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad).
7
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated and subjected to 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot. PVDF membranes were blocked in TBST solution with 5 % skim milk for 1 h at room temperature. After blocking, the membranes were probed with primary antibodies at room temperature for 2 h, which included KIF15 (1:2000, fine test, # FNab04551), AKT (1:2000, Proteintech, # 10176-2-AP), p-AKT (1:2000, Proteintech, # 66444-1-Ig), PI3K (1:2000, Proteintech, # 67071-1-Ig), p-PI3K (1:500, Bioss, bs-3332R) and GAPDH (1:3000, Bioworld, # AP0063). Following primary antibody incubation, membranes were exposed to Goat Anti-Rabbit (1:3000, Beyotime, # A0208) and Goat Anti-Mouse (1:3000, Beyotime, # A0216) secondary antibodies for 1 h at room temperature. After a series of washes with blocking solution, colorimetric detection was carried out using the ECL + plusTM western blotting system.
8
Protein Expression Analysis in Renal Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression levels of PI3K, p-PI3K, AKT, p-AKT, NFkB and p-NF-кB (p65) in renal tissues and cells of different groups were detected by western blot analysis according to our previous studies [19 (link)–21 (link)]. The detailed information about the antibodies is as follows: PI3K (#4249, CST, MA, USA), p-PI3K (#bs-3332R, Bioss Inc, Beijing, China), AKT (#ab89402, abcam, MA, USA), p-AKT (#ab81283, abcam, MA, USA), NF-кB (p65) (#YM3111, Immuno Way, TX, USA), p-NF-кB (p65) (#YP0191, Immuno Way, TX, USA), and GAPDH (# 2118, CST, MA, USA).
9
Western Blot Analysis of Exosomal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exosomes or cells were lysed with RIPA buffer, then the protein concentration was measured using the BCA protein detection kit (Beyotime, China). Each sample was mixed with protein loading buffer (5×) and heated at 95°C for 10 min to denature. Then protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Germany). PVDF membranes were blocked with 5% skim milk for 1 h, and primary antibody (MMP-13, collagenase II, IGFR1, p-PI3K, p-AKT, GAPDH, and β-ACTIN) (Bioss, China) was added in for incubating overnight at 4°C, then the secondary antibody conjugated with horseradish peroxidase was incubated for 2 h at room temperature. Fluorescence was analyzed using the enhanced chemiluminescence kit (Tanon, China) and imaged by the luminescent image analyzer (Tanon, China), and the absorbance value of each band was calculated using the ImageJ software.
10
Evaluating Anticancer Effects of B. coagulans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H22 cells (3 × 105cells/hole) in a logarithmic growth phase were plated in a 6-well plate and were incubated with B. coagulans MZY531 (MOI = 0, 50, 100) and 5-FU (100 µg/mL). After 24 h, the whole cell extract was prepared with RIPA buffer containing 1 mm PMSF. The protein concentration was detected by the BCA method, separated by SDS-PAGE electrophoresis, and transferred to the PVDF membrane. Western blot analysis was performed using the following primary anti-rabbit monoclonal antibodies: β-actin (bsm-52846R, BIOSS), PI3K (bs-10657R, BIOSS), p-PI3K (bs-5570R, BIOSS), AKT (bs-0115R, BIOSS), p-AKT (bs-0876R, BIOSS), mTOR (bsm-54471R, BIOSS), p-mTOR (bs-5331R, BIOSS), and Bax (GTX109683, GeneTex), caspase-3 (GTX110543, GeneTex) and Bcl-2 (GTX100064, GeneTex). The membrane was incubated with anti-rabbit second antibody conjugated with horseradish peroxidase at 37 ℃ for 1 h. the strips were quantified using image quant Las 4000 (Fuji film, Tokyo, Japan), and β-actin was used as a loading control.
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!