Total protein was extracted from small intestine tissues and enterocytes by lysis buffer for Western blotting with phenylmethanesulfonyl fluoride (PMSF) (100 mmol/L). These extracts were subjected to Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol at 4 °C. The membranes were blocked with 5% skim milk for 2 h and incubated overnight with diluted primary antibodies against Beclin 1 (1:500, Wanleibio, China), LC3 (1:500, ABclonal, China), mTOR (1:500, Wanleibio, China), AMPK (1:800, the polyclonal antibody produced by our lab), CAMKK-β (1:500, Proteintech, China), SERCA (1:500, the polyclonal antibody produced by our lab) and β-actin (1:10,000, ABclonal, China) followed by goat anti-rabbit IgG (H + L) (1:10,000, Immuno Way, China). The signal was detected using an enhanced chemiluminescence system (Zheng et al., 2020 (link)).
Beclin 1
Manufactured by Wanlei
Sourced in China
Beclin-1 is a protein involved in the regulation of autophagy, a cellular process that recycles and degrades damaged or unwanted cellular components. Beclin-1 plays a critical role in the initiation and formation of autophagosomes, which are the double-membrane vesicles that engulf cellular material for degradation.
Automatically generated - may contain errors
Lab products found in correlation
Cyclind1, by Wanlei (2 mentions)
β actin, by Wanlei (2 mentions)
β actin, by ABclonal (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
P erk, by Wanlei (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
Vimentin, by Wanlei (1 mentions)
Hoechst 3342, by Thermo Fisher Scientific (1 mentions)
C jun, by Wanlei (1 mentions)
Live dead assay kit, by Beyotime (1 mentions)
Goat anti rabbit igg hrp, by Wanlei (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
N acetyl cysteine (nac), by Beyotime (1 mentions)
Cleaved caspase 3, by Wanlei (1 mentions)
Bcl 2, by Wanlei (1 mentions)
Sp600125, by Beyotime (1 mentions)
Ros kit, by Beyotime (1 mentions)
Bcl2 associated x (bax), by Wanlei (1 mentions)
E cadherin, by Wanlei (1 mentions)
5 protocols using beclin 1
1
Western Blot Analysis of Intestinal Autophagy
2
Mitochondrial Protein Extraction and Analysis
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The radioimmunoprecipitation assay lysis buffer and phenylmethylsulfonyl fluoride were used to extract total protein. Tissue mitochondria isolation kit (Beyotime, C3606) was used to extract cytoplasm proteins (without mitochondria). The primary antibodies include β-actin (ABclonal, AC026), p53 (Bioss, bs-8687R), p-p53 (ABclonal, AP0083), p21 (ABclonal, AP0083), p16 (ABclonal, AP0083), Sirt3 (KleanAB, P100413), SOD2 (ABclonal, A19576), pink1 (ABclonal, A11435), Parkin (Wanleibio, WL02512), LC3 (Wanleibio, WL01506), p62 (ABclonal, A7758), Beclin1 (ABclonal, A7353), cGAS (ABclonal, A8335), STING (ABclonal, A21051), PERK (ABclonal, A18196), eIF2α (ABclonal, A21221), CHOP (Wanleibio, WL008800), TFAM (ABclonal, A13552), Sam50 (ABclonal, A3401), Mic60 (ABclonal, A2751), Mic19 (ABclonal, A8584), ATAD3A (ABclonal, A8230), and cytochrome c oxidase (COX) IV (ABclonal, A6564).
3
Peiminine Modulates Autophagy and Apoptosis
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Antibodies targeting the following proteins were used in this study: cyclin D1 (WL01435a), CDK2 (WL01543), p27 (WL04174), cleaved caspase-9 (WL01838), cleaved caspase-3 (WL01992), Bcl-2 (WL01556), Bax (WL01637), LC3BII/I (WL01506), beclin-1 (WL02508), p62 (WL02385), p-JNK (WL01813), JNK (WL01295), c-JUN (WL02863), E-cadherin (WL01482), vimentin (WL01960), MMP-2 (WL1579), MMP-9 (WL01580), β-actin (WL01372), goat anti-rabbit IgG-HRP (WLA023) (all from Wanleibio, China), and p-c-JUN (AF3095, Affinity, United States). Purified peiminine (98%) was obtained from Yuanye Bio Co., Ltd (Shanghai, China; B20081). Cell Counting Kit-8 (CCK-8; C0037), the live/dead assay kit, the cell cycle and apoptosis analysis kit, the ROS Kit, NAC, SP600125, and the terminal dUTP nick-end labeling (TUNEL) Kit (C1086) were acquired from Beyotime Institute of Biotechnology (Shanghai, China). Tetramethylrhodamine methyl ester (TMRM; I34361) and Hoechst 3342 (H3570) were sourced from Life Technology (OR, United States). The mRFP-GFP-LC3 adenovirus was acquired from HanBio Technology Co., Ltd (Shanghai, China). The rest of the reagents and experimental materials were purchased from common commercial sources.
4
Western Blot Analysis of Cell Cycle and Autophagy Markers
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FA was purchased from Meilunbio (Dalian Meilun Biotechnology Co., LTD. Liaoning, China). Antibodies for P53, P21, Cyclin D1, Cyclin E, Beclin-1, LC3-II, Atg12-Atg5 and β-actin used for Western blot analysis were purchased from Wanleibio (Shenyang, Liaoning, China). Super moloney-murine leukemia virus (M-MLV) reverse transcriptase for fluorescence quantification was purchased from BioTeke (Beijing, China) and RNA simple Total RNA Kit was purchased from TIANGEN (Beijing, China).
5
Amygdala Protein Expression Analysis
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Protein samples were acquired 24 h of post-surgery as mentioned above (SOD activity assay). 30 µg of amygdala tissues were combined with loading buffer (Cat # P0015, Beyotime) and boiled to separate the proteins using SDS-PAGE (12%) and transfer them to a PVDF membrane. Then the membrane was blocked for 2 h at a temperature of 25 ˚C using 5% skimmed milk. The PVDF membrane was exposed to primary antibodies polyclonal rabbit anti-Akt (Cat # WL0003b, 1:1000; Wanlei, Shenyang, China), polyclonal rabbit anti-phosphorylated Akt (Cat # WLP001a, 1:1000; Wanlei, Shenyang, China), and Beclin-1 (Cat # WL02508, 1:1000; Wanlei, Shenyang, China) for overnight incubation at 4 °C after three washes
(5 minutes each) with TBS-T. On the second day, a secondary antibody (HRP Conjugated A niPure Goat Anti-Rabbit IgG, Cat # WLA023, 1:2000; Wanlei, Shenyang, China) was incubated with the PVDF membrane for 1 h at 25 ˚C. Image Lab software (Version 6.0, Bio-Rad Laboratories, MD, USA) was used to assess the intensity of the targeted bands after incubation with ECL chemiluminescence solution (WLA006; Wanlei, Shenyang, China) for 5 min at 25 ˚C. The internal reference was GAPDH (1:1000, Cat # WL01114, 1000; Wanlei, Shenyang, China).
(5 minutes each) with TBS-T. On the second day, a secondary antibody (HRP Conjugated A niPure Goat Anti-Rabbit IgG, Cat # WLA023, 1:2000; Wanlei, Shenyang, China) was incubated with the PVDF membrane for 1 h at 25 ˚C. Image Lab software (Version 6.0, Bio-Rad Laboratories, MD, USA) was used to assess the intensity of the targeted bands after incubation with ECL chemiluminescence solution (WLA006; Wanlei, Shenyang, China) for 5 min at 25 ˚C. The internal reference was GAPDH (1:1000, Cat # WL01114, 1000; Wanlei, Shenyang, China).
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