Cells were fixed with cold methanol for 15 min, washed with PBST, blocked with 10% serum for 1 h at room temperature, and then incubated with rabbit polyclonal anti-LC3B (1:200) (2775, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The anti-rabbit-Alexa Fluor® 488 (Life Technologies, A-11008) was used as secondary antibody at 1:500 dilution for 1 h at room temperature, followed by the addition of DAPI (1 µg/mL) for 10 min. Samples were visualized by Zeiss LSM 710 confocal microscope (ZEISS, Oberkochen, Germany).
Anti lc3b
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany
Anti-LC3B is a primary antibody that recognizes the microtubule-associated protein 1 light chain 3B (LC3B), a widely used marker for autophagy. It can be used to detect and monitor autophagic processes in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti p62, by Cell Signaling Technology (43 mentions)
Anti beclin 1, by Cell Signaling Technology (36 mentions)
Anti gapdh, by Cell Signaling Technology (31 mentions)
Anti atg5, by Cell Signaling Technology (31 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (24 mentions)
Anti mtor, by Cell Signaling Technology (23 mentions)
Anti β actin, by Cell Signaling Technology (23 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (19 mentions)
Anti parp, by Cell Signaling Technology (18 mentions)
Anti akt, by Cell Signaling Technology (18 mentions)
Anti β actin, by Merck Group (18 mentions)
Anti atg7, by Cell Signaling Technology (16 mentions)
Anti β actin, by Santa Cruz Biotechnology (15 mentions)
Anti cleaved parp, by Cell Signaling Technology (14 mentions)
Anti caspase 3, by Cell Signaling Technology (14 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Anti bcl 2, by Cell Signaling Technology (13 mentions)
Anti gapdh, by Santa Cruz Biotechnology (11 mentions)
Anti p erk, by Cell Signaling Technology (11 mentions)
Pvdf membrane, by Merck Group (11 mentions)
334 protocols using anti lc3b
1
Immunofluorescence Localization of LC3B
2
Western Blot Analysis of Cell Signaling Proteins
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Protein sample preparation and western blot assay were performed as previously described (24 (link)). The primary antibodies used were: anti-MTMR3 (1:500; cat. no. 12443; Cell Signaling Technology, Inc.), anti-Cyclin D1 (1:1,000; cat. no. 60186-1-1g; ProteinTech Group, Inc.), anti-cyclin-dependent kinase 2 (1:1,000; CDK2; cat. no. 11026-2-AP; ProteinTech Group, Inc.), anti-p62 (1:1,000; cat. no. 19117-1-AP; ProteinTech Group, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-Cyclin E (1:1,000; cat. no. sc-247; Santa Cruz Biotechnology, Inc.), anti-Cyclin A (1:1,000; cat. no. sc-751; Santa Cruz Biotechnology, Inc.), anti-cell division cycle 25 A (1:1,000; cdc25A; sc-7157; Santa Cruz Biotechnology, Inc.), anti-microtubule associated protein 1 light chain 3 (LC3) A (1:500; cat. no. 4599; Cell Signaling Technology, Inc.), anti-LC3B (1:500; cat. no. 3868; Cell Signaling Technology, Inc.) and anti-GAPDH (1:20,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). Bound HRP-labeled secondary antibody (1:5,000; cat. no. SC-2005 or SC-2054; Santa Cruz Biotechnology, Inc.) was assayed by super ECL detection reagent (Pierce; Thermo Fisher Scientific, Inc.). Protein density of western blots was analyzed using ImageJ 1.51k software (National Institutes of Health).
3
Immunoblotting and Immunoprecipitation of Autophagy-Related Proteins
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Whole cell lysates were generated using 1× RIPA buffer supplemented with protease inhibitor. Proteins were immunoblotted with the antibodies anti-LC3B (#3868), beta actin (#4967), INPP4B (#14543), LAMP1 (#3243) from Cell Signaling, anti-vATPase V1H (sc-166227), and cathepsin B (sc-365558) from Santa Cruz. Immunoblotting and immunoprecipitation of VPS34 was performed using the following antibody: Rabbit monoclonal antibody for PI3 Kinase Class III (1:1000 for Western blot or 1:50 for immunoprecipitation, 4263, Cell Signaling). Representative western blots used for figure generation are listed for visualization (Fig. S9 ).
4
Detailed Western Blot Methodology
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Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).
5
Western Blot Analysis of Signaling Proteins
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Western blot was performed as previously described.3 (link) The anti-PARP, anti-p-β-catenin (S33/37/T41), anti-β-catenin, anti-c-Myc, anti-AKT1, anti-p-AKT1 (S473), anti-p-mTOR (S2448), anti-mTOR and anti-LC3B antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-SQSTM1/p62 antibody was from Abcam (Cambridge, UK). The densitometry of the immunoblots was performed with Image J software (NIH, Bethesda, MD)
6
Immunostaining of Retinal Pigment Epithelium
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Cryosections were used for immunostaining in order to visualize the RPE and detect the autophagosomes localization. 5% BSA (bovine serum albumin) was used to block non-specific bindings. Sections were then incubated overnight at 4 °C with primary antibodies: anti-RPE65 (Abcam, Cambridge, UK) (1:250 in 1% BSA) and anti-LC3B (Cell Signaling, Danver, CO, USA) (1:250 in 1% BSA) for retinal pigment epithelium and autophagosomes detection, respectively. Secondary antibodies were: anti-rabbit IgG conjugated to green fluorescent dye (Alexa Fluor 488, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for anti-LC3B and anti-mouse IgG conjugated to red fluorescent dye (Alexa Fluor 594, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for anti-RPE65. Secondary antibodies were diluted 1:1000 in Phosphate Buffered Saline (PBS 1X) and incubated at 37 °C for 2 h. Confocal images were acquired, by setting up the same parameters, using a Leica TCS SP5 confocal microscope.
7
Autophagy Protein Expression Analysis
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Western blot analysis was implemented using primary neuronal cells and SK-N-SH cells as described previously [40 (link)]. Primary antibodies used for immunoblotting were anti-LC3B (#4108, Cell Signaling Technology), anti-P62 (#5114, Cell Signaling Technology), anti-ATG5 (#2630, Cell Signaling Technology), and anti-β-actin (A5441, Sigma Aldrich).
8
Antibody Reagents for Cell Signaling
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Anti-LC3B, anti-Bcl-xL, anti-Atg16L1, anti-Rac1, anti-Rho1, anti-Cdc42, anti-phospho-ERK, anti-EEA1 and anti-LAMP1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies reactive with LRP1 (11H4) were a kind gift from Dr. Strickland, University of Maryland School of Medicine, Baltimore. Fibronectin (EP5) and ubiquitin (P4D1) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); anti-Cx43, anti-ERK, anti-Bcl-2 and anti-RPTPβ antibodies were from BD Biosciences (Tokyo, Japan); anti-multi ubiquitin monoclonal antibody (FK1) was from MBL (Nagoya, Japan); anti-GAPDH antibody was from GeneTex (Irvine, CA, USA) and anti-LC3 (clone 1703) antibody was from Cosmo Bio (Tokyo, Japan). Anti-RPTPα rabbit polyclonal antibodies for immunoblotting were provided by Dr. Jan Sap; anti-α-tubulin and anti-FLAG M2 antibodies, N-acetyl-l -cysteine (NAC) and ammonium chloride were from Sigma Aldrich (St. Louis, MO, USA); ProLong Gold Antifade Reagent with DAPI and DIDS were from Invitrogen (Eugene, OR, USA); an ERK inhibitor, U0126, was from Cayman Chemical (Ann Arbor, MI, USA); Rac1 inhibitor, NSC23766 was from Wako Pure Chemical Industries (Osaka, Japan); and GSH was from Merck (Darmstadt, Germany).
9
Lung Injury Assessment in Formalin-Fixed Tissue
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The collected right lung upper lobe was fixed with 10% neutral formalin, dehydrated, and embedded. The tissues were sliced into 4-µm-thick sections and stained with hematoxylin and eosin (HE). The stained tissues were photographed under an optical microscope (Nikon, Tokyo, Japan). The lung injury was scored by a histologic scoring system as described in a previously published article [11 (link)].
For immunofluorescence, the dewaxed and dehydrated sections were washed with 0.01 M PBST three times (5 min per time). After blocking with 10% BSA for 30 min at 37 °C, sections were incubated with anti-LC3B (1:50, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Cell nuclei were stained with DAPI. ImageJ software was used to count the LC3B-positive puncta per cell.
The left lung was used for BALF collection and the lung wet-to-dry ratio was calculated. The dry lung weight was recorded after incubation of the wet lung tissues for 72 h at 70 °C.
For immunofluorescence, the dewaxed and dehydrated sections were washed with 0.01 M PBST three times (5 min per time). After blocking with 10% BSA for 30 min at 37 °C, sections were incubated with anti-LC3B (1:50, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Cell nuclei were stained with DAPI. ImageJ software was used to count the LC3B-positive puncta per cell.
The left lung was used for BALF collection and the lung wet-to-dry ratio was calculated. The dry lung weight was recorded after incubation of the wet lung tissues for 72 h at 70 °C.
10
Multimodal Cancer Treatment Investigation
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The 5-fluorouracil, metformin, and ICG-001 used were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Selleckchem (Houston, TX, USA) respectively. The antibodies, such as anti-PARP, anti-cleaved PARP, anti-caspase 3, anti-cleaved caspase 3, anti-LC3B, anti-ATG9A, anti-CD44, anti-β-catenin, anti-OCT4, anti-β-actin, anti-p-AMPK, and anti-AMPK were procured from Cell Signaling Technology (Danvers, MA, USA). APC conjugated anti-CD44 was procured from BioLegend (San Diego, CA, USA). All the primers were procured from Integrated DNA Technologies (San Diego, CA, USA).
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