BV2 microglia were washed rapidly with ice-cold PBS, scraped, and collected. Cell pellets were lysed with ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). The lysates were centrifuged at 12,000 rpm for 30 min at 4℃ to produce whole-cell extracts. Protein content was quantified using the BCA method (Pierce, Rockford, IL, USA). Proteins (40 µg) were separated on a 10% SDS-polyacrylamide (PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin, prepared in Tris-buffered saline/Tween (TBS-T; 20 nM Tris [pH 7.2], 150 mM NaCl, and 0.1% Tween 20), for 1 h at room temperature, immunoblots were incubated overnight at 4℃ with primary antibodies that specifically detect BECLin-1 (1 : 1,000, Millipore, Billerica, MA, USA), ATG3 (1 : 1,000, Abcam, Cambridge, MA, USA), LC3II (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), and β-actin (1 : 2,000, Cell Signaling Technology, Danvers, MA, USA). Next, blots were incubated with HRP-linked anti-rabbit IgG antibodies purchased from Abcam (Abcam, Cambridge, MA, USA) for 1 h 30 min at room temperature. Enhanced chemiluminescence was performed by ECL (Invitrogen, Carlsbad, CA, USA) [49 (link)].
Beclin 1
Manufactured by Merck Group
Sourced in United States, China, United Kingdom
Beclin-1 is a core autophagy protein that plays a crucial role in the regulation of autophagy, a cellular process involved in the degradation and recycling of damaged or unwanted cellular components. It serves as a key regulator in the initiation and formation of autophagosomes, the double-membrane vesicles that engulf the target cargo for degradation. Beclin-1 is an essential component in the autophagic pathway and is involved in various cellular processes, including cellular homeostasis, stress response, and disease pathogenesis.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (9 mentions)
Bcl 2, by Cell Signaling Technology (5 mentions)
Lc3 2, by Merck Group (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Gapdh, by Merck Group (4 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Lc3 2, by Cell Signaling Technology (3 mentions)
Bcl 2, by Merck Group (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Rapamycin, by Merck Group (2 mentions)
3 methyladenine 3 ma, by Merck Group (2 mentions)
α tubulin, by Merck Group (2 mentions)
Bcl2 associated x (bax), by Merck Group (2 mentions)
Ripa buffer, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Parkin, by Santa Cruz Biotechnology (1 mentions)
Hsp60, by BD (1 mentions)
Mthsp70, by Thermo Fisher Scientific (1 mentions)
35 protocols using beclin 1
1
Western Blot Analysis of Autophagy Markers
2
Mitochondrial Signaling Pathways Analysis
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Antibodies against PGC1α (Ab3243; Millipore, Billerica, MA), transcription factor A, mitochondrial (TFAM; Abcam Ab119684), NEF2L2 (Abcam ab31163), ClpP (Sigma HPA010649), Htra2 (AF1458; R&D Systems, Minneapolis, MN), mtHsp70 (Thermo Fisher Scientific), Hsp60 (611562; BD Biosciences, Franklin Lakes, NJ), GPS2,21 p62 (Progen GP62‐C), Beclin1 (Millipore Ab15417), Pink1 (Abcam Ab23707), LC3‐II (4108; Cell Signaling Technology, Danvers, MA), and parkin (sc‐32282; Santa Cruz Biotechnology, Santa Cruz, CA) to assess retrograte signaling factors and mitophagy markers were used in combination with MTCOI, SDHA, and DAPI to assess changes in relation with COX deficiency in single muscle sections. Antibodies that did not produce a sufficient signal‐to‐noise ratio, or were highly correlated with mitochondrial mass, were removed from further analysis. TFAM, Hsp60, GPS2, and Beclin1 were selected for immunofluorescence on serial sections (n = 4) of patients with multiple mtDNA deletions (n = 3, Patients 4, 5, and 7; see Supplementary Table). IMARIS v.7.7.2 (Bitplane) was used to assess average fluorescent intensity of signaling markers relative to MTCOI/SDHA ratio in whole COX‐positive and COX‐deficient fibers. Average intensity of the foci was compared to a COX‐positive region of the fiber.
3
Autophagosome Formation in Colonic Macrophages
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The formation of autophagosomes in colonic macrophages after TNBS induction with or without IL-33 administration was compared by immunofluorescence assay. Briefly, the sections were deparaffinized, rehydrated and washed in 1% PBS Tween. Then they were treated with 3% hydrogen peroxide, blocked with 5% bovine serum albumin (BSA) and incubated simultaneously with Beclin-1 (1:500, Sigma) and F4/80 (1:500, Abcam) overnight. After staining with the- secondary antibody, the slides were then counter-stained with DAPI for 5 min. Images were acquired by a fluorescence microscope (Olympus, Lake Success, NY). Settings for image acquisition were identical for control and experimental tissues.
4
Molecular Mechanisms of Autophagy Regulation
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Cell culture reagents were purchased from Invitrogen. Human insulin-like growth factor 1 (hIGF-1) was purchased from Cell Signaling Technology (Boston, MA, USA). Chloroquine and U0126 were from Promega Corporation (Madison, WI, USA). The MG132 was from Sigma-Aldrich (Louis, MO, USA). Lambda protein phosphatase (λ-PP) was from New England BioLabs Inc. (Ipswich, MA, USA). The following antibodies were used throughout whole study. Rabbit polyclonal antibody β-Actin (P30002, Abmart, China), rabbit polyclonal antibody LC3 (L7543, Sigma, USA), rabbit monoclonal antibody Bim (2933, Cell Signaling Technology, USA), rabbit polyclonal antibody phosphorylated ERK1/2 (9101, Cell Signaling Technology, USA), rabbit polyclonal antibody native ERK1/2 (9102, Cell Signaling Technology, USA), rabbit polyclonal antibody Beclin1 (B6061, sigma, USA), rabbit polyclonal antibody Bcl-2 (2870, Cell Signaling Technology, USA), and rabbit polyclonal antibody Bax (AF0057, Beyotime, China), as well as goat anti-rabbit second antibody, HRP (31460, Thermo Fisher Scientific, USA). All other chemicals were purchased from Sigma-Aldrich, unless otherwise stated.
5
Investigating Solamargine-induced Autophagy
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Solamargine was purchased from TargetMol (batch:117797, purity: 99.76%, Boston, MA, USA), whereas BTZ was provided by MedChemExpress (Shanghai, China). 3‐MA was from Sigma (St Louis, MO, USA). RPMI 1640 medium and fetal bovine serum were from Gibco (Rockville, MD, USA). CCK‐8 was supplied by Vazyme (Nanjing, China). The following antibodies were used: LC3, Beclin 1 and β‐actin (Sigma), Bcl‐2, Bax, cleaved‐caspase‐3 and Ki‐67 (Cell Signalling Technology, Beverly, MA, USA), goat anti‐rabbit IgG‐horseradish peroxidase (HRP) and goat anti‐mouse IgG‐HRP (Pierce, Rockford, IL, USA). Other chemicals were of analytical grade and were purchased from local commercial sources.
6
Salvia and Pueraria Osteoprotective Effects
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Salvia miltiorrhiza Bunge and Radix Puerariae were purchased from Changda Prepared Chinese Medicinal Herbs Co. Ltd (Anguo, China). Healthy SD rats, SPF clean grade, female, 10 weeks old, weight 200–250 g, were purchased from Chinese Academy of Sciences; The ELISA kits for detecting blood urea nitrogen (BUN), creatinine, ALP, OPG and RANKL were purchased from Wuhan Huamei Biological co., LTD (Wuhan, China); DCFH2-DA, Fluo-3/AM, BAPTA-AM and Hoechst 33,258 were obtained from Molecular Probes (Eugene, OR); Chemicals used for DPI, TTRA, AA, ALL, NAC, NDGA, Rot were purchased from Sigma-Aldrich (St. Louis, MO). H&E and Masson dying kits were purchased from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China); 3-methyladenine (3-MA), and monodansyl cadaverine (MDC) were obtained from MedChemExpress; TRAP staining kit was obtained from BIO-SCIENCE COMPANY LIMITED (Shanghai, China); Antibodies GAPDH, LC3B, Beclin-1, p62, p-p65 and NF-κBp65 were purchased from Sigma Company; Antibodies NFATc1 and c-Fos were purchased from Chengdu Zen Bio (Chengdu, China); Secondary antibodies were purchased from American CST Company; Hoechst 33,342 was purchased from Thermo Fisher Scientific; JYB1-1 calcium removal solution and protein extraction kit were purchased from Solarbio Company (Beijing, China); RANKL was purchased from Sino Biological Company (Beijing, China).
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Salvia miltiorrhiza Bunge and Radix Puerariae were purchased from Changda Prepared Chinese Medicinal Herbs Co. Ltd (Anguo, China). Healthy SD rats, SPF clean grade, female, 10 weeks old, weight 200–250 g, were purchased from Chinese Academy of Sciences; The ELISA kits for detecting blood urea nitrogen (BUN), creatinine, ALP, OPG and RANKL were purchased from Wuhan Huamei Biological co., LTD (Wuhan, China); DCFH2-DA, Fluo-3/AM, BAPTA-AM and Hoechst 33,258 were obtained from Molecular Probes (Eugene, OR); Chemicals used for DPI, TTRA, AA, ALL, NAC, NDGA, Rot were purchased from Sigma-Aldrich (St. Louis, MO). H&E and Masson dying kits were purchased from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China); 3-methyladenine (3-MA), and monodansyl cadaverine (MDC) were obtained from MedChemExpress; TRAP staining kit was obtained from BIO-SCIENCE COMPANY LIMITED (Shanghai, China); Antibodies GAPDH, LC3B, Beclin-1, p62, p-p65 and NF-κBp65 were purchased from Sigma Company; Antibodies NFATc1 and c-Fos were purchased from Chengdu Zen Bio (Chengdu, China); Secondary antibodies were purchased from American CST Company; Hoechst 33,342 was purchased from Thermo Fisher Scientific; JYB1-1 calcium removal solution and protein extraction kit were purchased from Solarbio Company (Beijing, China); RANKL was purchased from Sino Biological Company (Beijing, China).
7
Signaling Pathways in Cardiac Hypertrophy
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Antibodies were obtained from the following companies: GAPDH, ERK, p-ERK, MEK, p-MEK, beclin-1, ANP, BNP, Noxa, LC3-I, LC3-II, Mcl-1, P62, mTOR, p-mTOR, Akt, p-Akt, P70S6K1, and p-P70S6K1 (Sigma, St. Louis, MO), Rapamycin and phenylephrine (PE) (Abcam, Cambridge, USA). DMEM (high glucose) and fetal bovine serum were from Nego (Shanghai, China). Cell lysis buffer (10 ×) was obtained from Cell Signaling Technology (Massachusetts, USA). The RT-PCR kit was purchased from TOYOBO (Shanghai China). Other reagents included DAPI (Roche, Germany), hematoxylin and eosin (H&E, Toronto Chemicals, Toronto, Canada), and trypsin (Sigma). GFP-LC3 was purchased from Biovector Science Lab, Inc. (Beijing, China). All pairs of real-time PCR primers were synthesized by Shenggong Biotechnology (Shanghai, China). Other chemicals and reagents were of analytical grade.
8
Immunofluorescence Analysis of Autophagy Markers
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Liver tissue was perfused and fixed, and 10-µm-thick frozen sections were prepared, fixed in 4% paraformaldehyde for 15 min, and washed with PBS. The endogenous peroxidase was inactivated by blocking with 1% goat serum for 30 min at room temperature and then incubated overnight at 4 °C with diluted primary antibody (LC3-II, 1:200, Sigma; Beclin1, 1:200, Sigma; ATG-7, 1:200, Sigma; P62, 1:200, Sigma). The following day, it was washed three times with PBS, incubated with the secondary antibody for 60 min at room temperature, washed three times with PBS, and nucleus were stained by DAPI (1 mg/mL), then observed under the fluorescence microscope (Olympus, Japan).
9
Autophagy and Apoptosis Markers at 14 Months
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We measured markers of autophagy and apoptosis at 14 months by previously described western. Primary antibodies to Beclin-1 (#PRS3613, Sigma-Aldrich), Caspase-3 (#9662, cell signaling), BCL-2 (#2570, cell signaling), p62 (#8025, cell signaling) and LC3 (#NB100-2220, Novus) were used with HRP-conjugated secondary antibodies (GE lifesciences).
10
Western Blot Analysis of Protein Signaling
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Western blot analysis was performed as previously described8 (link). In brief, total protein samples extracted from untreated and treated tissues or cells were prepared using RIPA buffer containing a protease inhibitor cocktail (Roche, Branford, CT, USA) and a phosphatase inhibitor and were subjected to SDS PAGE (90μg protein per sample). The membranes were then incubated with the following primary antibodies: p-IRS-1 (Cell Signaling Inc. (CST), Danvers, MA, USA,1:800), eNOS (CST,1:1,000), p-eNOS (Thermo Fisher,1:800), IRS-1 (CST,1:1,000), p-AKT (CST,1:1000), AKT (CST,1:1000), p-PI3K (Abcam, Cambridge, MA, USA,1:500), PI3K(Abcam1:1000),GAPDH (CST, 1:1,500), anti-LC3 (1:200, Sigma-Aldrich, St. Louis, MO, USA), Beclin1 (1:200, Sigma-Aldrich), GLUT4 (1:3,000, Thermo Fisher), p-JAK2 and JAK2(CST,1:1000).
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