Cells were seed on glass bottom dishes (SORFA Life Science, Beijing, China) and fixed with 4% paraformaldehyde (w/v) after treatment at room temperature for 30 min. Then the cells were permeabilized with 0.1% Triton X-100 for 10 min after washing with PBS for three times. The samples were blocked with 5 % BSA at room temperature for 30 min and incubated with primary antibodies at 4 °C overnight: Olig2 (Abcam, Cat# ab109186), Iba1(Abcam, Cat# ab178846), GFAP (Abcam, Cat# ab68428), NeuN (Abcam, Cat# ab177487), LAMP2 (Proteintech, Cat# 66301-1-Ig), TFEB (Proteintech, Cat# 13372-1-AP), Annexin A7 (Proteintech, Cat# 10154-2-AP), LAMP5 (Santa Cruz, Cat# sc-398190), LC3B (Abcam, Cat# ab192890), Cleaved-caspase3 (CST, Cat# 9661 S). Samples were incubated with secondary antibodies at 37 °C for 1 h after washing with TBST for three times. Finally, samples were stained with DAPI for 10 min to show nucleus, and fluorescence signals were detected by laser scanning confocal microscopy (Zeiss LSM900, Germany).
Lamp2
Manufactured by Proteintech
Sourced in China
LAMP2 is a protein that plays a role in the process of autophagy, which is the degradation and recycling of cellular components. It is a member of the lysosome-associated membrane protein (LAMP) family and is involved in the formation and maturation of autophagosomes, which are the vesicles that engulf cellular material for degradation.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (3 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Olig2, by Abcam (1 mentions)
Annexin a7, by Proteintech (1 mentions)
Lamp5, by Santa Cruz Biotechnology (1 mentions)
Lsm 900, by Zeiss (1 mentions)
Neuronal nuclei (neun), by Abcam (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Annexin 5 fitc pi kit, by Keygen Biotech (1 mentions)
Cell cycle detection kit, by Keygen Biotech (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Trizol reagent, by CoWin Biotech (1 mentions)
Ultrasybr mixture, by CoWin Biotech (1 mentions)
3 methyladenine 3 ma, by MedChemExpress (1 mentions)
Parp1, by Proteintech (1 mentions)
Cdna synthesis kit, by CoWin Biotech (1 mentions)
Ca 074 methyl ester ca 074 me, by MedChemExpress (1 mentions)
Lyso tracker red dye, by Beyotime (1 mentions)
10 protocols using lamp2
1
Immunofluorescence Staining of Cell Cultures
2
Evaluation of UA232 in ER Stress-Induced Apoptosis
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UA232 was synthesized in our laboratory with a purity of > 98%. UA, 3-methyladenine (3-MA), and CA-074 methyl ester (CA-074-me) were purchased from MedChemExpress (Monmouth Junction, NJ, USA), and 4-phenylbutyric acid (4-PBA) was purchased from Energy Chemical (Shanghai, China). Crystal violet, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), 4% paraformaldehyde fix solution, Lyso-Tracker Red dye, and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Biotechnology (Shanghai, China). The cell cycle detection kit and Annexin V-FITC/PI kit were purchased from KeyGen Biotech (Nanjing, China). Primary antibodies against cyclin D1, CDK4, Bax, Bcl-2, caspase-8, caspase-3, PARP1, CHOP, GRP78, ATF4, LC3, P62, and LAMP2 were purchased from Proteintech Group (Rosemont, IL, USA). Antibodies against PERK, p-PERK, eIF2a, and p-eIF2a were purchased from Bioss (Beijing, China). Antibodies against cathepsin B were purchased from Abcam (Cambridge, MA, USA). β-actin, horseradish peroxidase conjugated secondary antibodies (goat anti-mouse or goat anti-rabbit), and fluorescently labeled secondary antibodies (IgG Alexa Fluor® 488 or 594 goat-anti-rabbit) were obtained from ZSGB BIO (Beijing, China). Trizol reagent, cDNA synthesis kit, and UltraSYBR mixture were purchased from CoWin Biotech (Beijing, China).
3
Protein Extraction and Western Blot Analysis
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After desired treatments as specified, cells were washed twice with PBS and lysed in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycer-ophosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) on ice. Equal amounts of protein (30 μg) were loaded onto 8% SDS-PAGE gels. Western detection was performed using a chemiluminescence Western blot scanner (ChampGel 7000, SAGECREATION). For protein detection, the following antibodies were used: ACC1 (#21923-1-AP; Proteintech), ACC2 (#8578; Cell Signaling), phosphorylation ACC (#3661; Cell Signaling), CD36 (#18836-1-AP; Proteintech), LAMP2 (#66301-1-Ig; Proteintech), TOM20 (ab56783; Abcam), GAPDH (#60004-1-Ig; Proteintech), Lamin B1 12987-1-AP(proteintech), RAC1 (#24072-1-AP; Proteintech), β-Actin (#AC026; Abclonal), β-tubulin (#AC008; Abclonal), goat anti-rabbit IgG/HRP (#115-035-003; Jackson) and goat anti-mouse IgG/HRP (#111-035-003; Jackson).
4
Western Blotting of Autophagy Proteins
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Cell lysates were prepared using lysis buffer composed of 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40. The lysates were centrifuged to collect the supernatants. An equal amount of protein was denatured in SDS sample buffer and separated on 8% or 10% polyacrylamide gels based on the molecular weight of the target proteins. The separated proteins were then transferred to a PVDF membrane. The membranes were blocked with 5% nonfat milk in TBST (TBS containing 0.05% Tween 20), incubated with primary antibodies, and subsequently incubated with secondary antibodies conjugated to alkaline phosphatase. Protein expression was detected by chemiluminescence. The antibodies used were against ALKBH5 (ab195377, Abcam, Britain), ZKSCAN3 (ab223477, Abcam, Britain), LC3 (Proteintech, Wuhan, China), LAMP-2 (Proteintech, Wuhan, China) and SQSTM1 (Proteintech, Wuhan, China).
5
Optimizing Autophagy Modulation for Cancer Therapy
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The reagents used in this study were purchased from Chinese suppliers: Z-VAD-fmk (CSNpharm, CSN15936, Chicago, USA), CQ (Sigma-Aldrich, C6628, St. Louis, MO, USA), NAC N-acetyl-L-cysteine (Beyotime, S0077, Shanghai, China), AO (Sigma-Aldrich, A6014), 3-MA (Selleck, S2767, Houston, Texas, USA), rapamycin (Selleck, S1039), Cisplatin (Selleck, S1166), and Dorsomorphin (Selleck, S7846). The following antibodies were also purchased from Chinese suppliers: GAPDH (Proteintech, 10,494–1-AP, Wuhan, China), cathepsin D (Proteintech, 21,327–1-AP), PCNA (Proteintech, 10,205–2-AP), Ki-67 (Proteintech, 27,309–1-AP), LAMP2 (Proteintech, 66,301–1-lg), p70s6K (Proteintech, 14,485–1-AP), tubulin (Proteintech,1124–1-AP), P21(Proteintech, 10,355–1- AP), cleaved-caspase9 (Asp175) (CST, 52873, Beverly, MA, USA), cleaved caspase 8 (Asp391) (CST,9496), Bax (CST 2774), Bid (CST,2003), Raptor (CST,2282), p-raptor(Ser792) (CST,2083), Rab7 (CST,9367), AMPK (CST, 5832), p-AMPK (CST, 2535), mTOR (CST, 2983), p-mTOR (CST,5536), p-p70s6K (CST,9234), LC3B (CST, 3868), LAMP1 (CST 15665), Atg7 (CST,8558), p62 (CST, 88588).
6
Protein Extraction and Western Blot Analysis of Mouse Lung Tissues
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Total protein was extracted from mouse lung tissues and cells using a prepared SDS lysis buffer for protein blotting analysis. The proteins were separated on SDS–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). The membrane was then blocked with 5% skimmed milk for 2 h. Antibodies against β-actin (Abcam, #ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), TGF-β1 (ImmunoWay, #YT4632), P-NFKB (Wanleibio, #WL02169), NFKB (Bioss, #bsm-33117M), TNF-α (Proteintech, #60291-1-Ig), IL-1β (Wanleibio, #WLH3903), HIF-1a (Proteintech, #20960-1-AP), SOD1 (Wanleibio, #WL01846), SOD2 (Boster, #BA4566), LC3-II (Cell Signaling Technology, #12741), P62 (Cell Signaling Technology, #16177S), LAMP2 (Proteintech, #66301-1-Ig), P-mTOR (Abmart, #T56571), mTOR (Abmart, #T55306), P-ULK1 (ImmunoWay, #YP1544), ULK1 (Abmart, #T56902), Beclin1 (Boster, #PB0014), Atg5 (Bioss, #bs-4005r), BNIP3 (Santa Cruz Biotechnology, #sc-56167), P-AKT (Abmart, #T40067), AKT (Abmart, #BM4400), and TMEM175 (Proteintech, #19925-1-AP) were used. The PVDF membrane was incubated with these antibodies at 4 °C overnight. Subsequently, an HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, followed by detection using the ECL luminescence system.
7
Immunofluorescence Staining of LC3 and LAMP-2
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After cell fixation, the cells were treated with 0.2% Triton X-100 at room temperature. The cells were then blocked with blocking solution. Subsequently, the cells were treated with primary and secondary antibodies. DAPI dye was added to the cells, which were subsequently incubated in the dark. The cells were mounted on slides using anti-fade mounting medium, and fluorescence was observed using a fluorescence microscope. The antibodies used were against LC3 (Proteintech, Wuhan, China) and LAMP-2 (Proteintech, Wuhan, China).
8
Antibody Labeling and Protein Detection
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Antibodies against the following proteins were used in this study: TH (abcam, ab137869; 1:5000), Trx-1 (Proteintech, 14999-1-AP; 1:2000), α-syn (abcam, ab212184; 1:2000), PINK1 (Santa Cruz Biotechnology, sc-517353; 1:500), Parkin (Santa Cruz Biotechnology, sc-32282; 1;500), LC3I/II (Cell Signaling Technology, #4108; 1:1000), p62 (Proteintech, 66184-1-Ig; 1:2000), cathepsin B (Proteintech, 12216-1-AP; 1:2000) cathepsin D (Santa Cruz Biotechnology, sc-377299; 1:500), LAMP2 (Proteintech, 66301-1-Ig; 1:2000), β-actin (Proteintech, 81115-1-RR; 1;10000), Anti-Mouse IgG (H + L) Antibody, (SeraCare, 5450-0011; 1:10000), Anti-Rabbit IgG (H + L) Antibody (SeraCare, 5450-0010; 1:10000). Fluorophore-conjugated secondary antibodies were as follows: Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (Thermo Fisher, A-11036; 1:1000). Other reagents included MPTP-HCl (Sigma-Aldrich-Corporation, 23007-85-4), MPP+ (Sigma-Aldrich-Corporation, 36913-39-0), Rapamycin (MedChemExpress, HY-10219), Chloroquine phosphate (MedChemExpress, HY-17589), Antifading Mounting Medium with DAPI, (Solarbio, S2110).
9
Protein Extraction and Western Blot Analysis
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Protein extraction and WB assays were performed as previously described [28 (link)]. Primary antibodies targeting the following proteins were used in our WB analysis: Lamp2 (Proteintech, Wuhan, China, 27823-1-AP, 1:2000), EEA1 (Proteintech, 28347-1-AP, 1:2000), LC3B (Proteintech, 18725-1-AP, 1:2000), CD63 (Abcam, Burlingame, CA, USA, ab134045, 1:2000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), NOS3 (Proteintech, 27120-1-AP, 1:2000), VAMP3 (Proteintech, 10702-1-AP, 1:2000), TSG101 (Proteintech, 28283-1-AP, 1:2000), Calnexin (Proteintech, 10427-2-AP, 1:2000).
10
Antibodies and Compounds for Ferroptosis Study
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The primary antibodies for SLC7A11 (12691 (Cell Signaling Technology), ab175186 (Abcam) and 26864‐1‐AP (Proteintech), Flag tag (14793; Cell Signaling Technology), ACSL4 (ab155282; Abcam), Streptavidin (3419; Cell Signaling Technology), GPX4(ab125066; Abcam), LAMP2 (66301‐1‐Ig; Proteintech), Anti‐4Hydroxynonenal (ab48506; Abcam), GAPDH (ab8245; Abcam), andβ‐actin (ab8226; Abcam) were commercially available. The following secondary antibodies were used in immunofluorescence assays: AF488‐anti‐Mouse (Invitrogen), AF594‐anti‐rabbit (Invitrogen), AF594‐anti‐mouse (Invitrogen), AF568‐anti‐rabbit (Abcam), AF647‐anti‐rabbit (Abcam), and AF647‐anti‐mouse (Abcam). Small‐molecular compounds such as Sorafenib (HY‐10201), Erastin (HY‐15763), Bafilomycin (HY‐100558), MG132 (HY‐13259), Cycloheximide (HY‐12320), Chlorquinaldol (HY‐17589A), Z‐VAD‐FMK(HY‐16658B), Necrosulfonamide (HY‐100573) and Ferrostatin‐1 (HY‐100579) were all from MCE. 2‐BP (21604) was from Sigma–Aldrich.
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