PLGA (obtained from Dai Gang, Beijing Shi, China; molecular weight [Mw] =5×105 Da, l -lactide/glycolide ratio =75:25) was used as the scaffold polymer. N,N-dimethylformamide ([DMF]; obtained from the Chinese Medicine Group, SINO-PHARM, Beijing, China) was used as the solvent. DMEM and horse serum (HS) were purchased from Hyclone (Logan, UT, USA). Penicillin (100 units/mL), streptomycin (100 mg/mL), and trypsin-EDTA solution were purchased from Beyotime (Shanghai, China). FBS, ammonium persulfate, and PBS were purchased from Grand Biological Technology Co., Ltd (Shijiazhuang, China). Specific primary antibodies for glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH), myosin heavy chain (MyHC), and myogenin (MyOG) were purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies (horseradish peroxidase [HRP]-conjugated anti-rabbit or anti-mouse) were purchased from TransGene Biotech (Beijing, China). An ECL-plus kit was purchased from Advansta (MenloPark, CA, USA). Nonfat milk was purchased from Difco (Franklin Lakes, NJ, USA). Triton X-100, tetramethylethylenediamine, and N,N-methylene bisacrylamide were obtained from Sigma (Shanghai, China). Alexa Fluor 568-Phalloidin and ProLong Gold Antifade Reagent, paraformaldehyde, and DAPI were purchased from Biotopped Science & Technology Co. Ltd. (Beijing, China).
Ecl plus kit
Manufactured by Advansta
Sourced in United States
The ECL-plus kit is a chemiluminescent detection system used for the quantitative analysis of proteins in Western blot applications. It provides a sensitive and reliable method for detecting target proteins labeled with specific antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Triton x 100, by Merck Group (1 mentions)
N n methylenebisacrylamide, by Merck Group (1 mentions)
Tetramethylethylenediamine, by Merck Group (1 mentions)
Streptomycin, by Beyotime (1 mentions)
Penicillin, by Beyotime (1 mentions)
Trypsin edta solution, by Beyotime (1 mentions)
Myogenin myog, by Cell Signaling Technology (1 mentions)
Polybrene, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Antibodies against α smooth muscle actin α sma, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
P ulk1, by Cell Signaling Technology (1 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Nvp bez235, by Selleck Chemicals (1 mentions)
Cck8 kit, by 7Sea Biotech (1 mentions)
Nonfat milk, by BD (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
6 protocols using ecl plus kit
1
PLGA-based Scaffold Fabrication
2
Signaling Pathway Profiling via Western Blot
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Primary antibodies against total-p65, phospho-p65 (S536), total-c-Jun, phospho-c-Jun (Ser63/Ser73), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technologies (Danvers, MA, U.S.A.). Antibodies against NMBR weres purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Antibodies against α-smooth muscle actin (α-SMA) were purchased from Abcam (Cambridge, U.K.). Horseradish peroxidase (HRP)–conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Cell Signaling Technologies (Danvers, MA, U.S.A.). An ECL-plus kit was purchased from Advansta (MenloPark, CA, U.S.A.). NMB was purchased from Bachem (Bubendorf, Switzerland). The human IL-6 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Enzo Life Sciences (New York, NY, U.S.A.). Polybrene was purchased from Invitrogen (San Diego, CA, U.S.A.).
3
Investigating Autophagy and Apoptosis Pathways
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Primary antibodies against ULK1, p-ULK1, AMPK, p-AMPK, LC3-II, cleaved-caspase3 and Actin were purchased from Cell Signaling Technology (CST). HRP-conjugated anti-rabbit or anti-mouse secondary antibodies and an ECL-plus kit were from Advansta in America. Chloroquine (CQ) was purchased from Sigma-Aldrich and NVP-BEZ235 was purchased from selleck. CCK-8 kit was from 7 sea biotech (Shanghai, China).
4
Western Blot Analysis of Protein Expression
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Cells were extracted with RIPA lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail (Roche, Basel, Switzerland). Protein lysates were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk (BD Bioscience) for 2 h at room temperature, the membranes were incubated with one of the following primary antibodies: anti-DDDDK tag (ab49763, Abcam, Cambridge, MA, USA), LDHA (ab101562, Abcam), phospho-LDHA (Tyr10) (8176, Cell Signalling Technology, Danvers, MA, USA), cyclin G2 (HPA034684, Sigma-Aldrich) or GAPDH (5174, Cell Signalling Technology) overnight at 4°C. The membranes were incubated with an HRP-linked secondary antibody (anti-rabbit IgG; Thermo Fisher Scientific, Waltham, MA, USA) for 2 h. Proteins of interest were visualised using an ECL Plus kit (Advansta, Menlo Park, CA, USA). Three independent experiments were performed.
5
Western Blot Analysis of Cell Signaling
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After drug treatment for indicated time, cell protein samples were extracted with radioimmunoprecipitation assay (RIPA) buffer [10 mmol/L Tris‐Cl (pH 8.0), 1 mmol/L EDTA, 0.5 mmol/L EGTA, 1% Triton X‐100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mmol/L NaCl]. Equivalent protein samples (30 μg protein extract was loaded on each lane) were separated by 10% SDS‐PAGE, transferred onto PVDF membranes (Millipore) and blocked with 5% nonfat milk for 1 hour at room temperature. The membranes were incubated with anti‐p53, p73, phospho‐Akt (S473), total‐Akt, PUMA, phospho‐FoxO3a, total‐FoxO3a, phospho‐p65 (S276 and S536), total‐p65 and cleaved caspase3 (Cell Signaling Technology), and β‐actin antibody (Santa Cruz) primary antibodies at 4°C overnight. Primary antibody was detected by binding horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse secondary antibody with an ECL plus kit (Advansta, MenloPark, CA, USA). β‐actin was used as a loading control.
6
Comprehensive Protein Analysis in Research
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Primary antibodies against LC3, Poly-ADP-ribose polymerase-1 (PARP-1), CD161c (NK1.1) were purchased from Cell Signaling Technology (Boston, Massachusetts, USA); N-cadherin, E-cadherin, Vimentin, P62, CRT, HSP-70, HSP-90, GPX-4, Ki67, PD-L1, Bax and β-actin were purchased from Proteintech (Wuhan, China); poly-ADP ribose (PAR) and Anti-cleaved N-terminal Gasdermin-D (N-GSDMD) were purchased from Abcam (Cambridge, UK); CD8a was purchased from Thermo Fisher (Waltham, Massachusetts, USA); CD4, CD11c, F4/80, CD86 and CD206 were purchased from CUSABIO (Houston, USA). Horseradish peroxidase (HRP) conjugated anti-rabbit/mouse secondary antibodies were purchased from Abbkine (Wuhan, China) and the ECL-plus kit was from Advansta in USA. Cell Counting Kit-8 (CCK-8) was from 7 Sea Biotech (Shanghai, China).
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