Immunofluorescence was performed according to method in use in the laboratory [34 (link)]. Cells were fixed with absolute alcohol, washed, and, blocked with 3% bovine serum albumin in PBS for 30 min. The cells were then incubated overnight with anti-Vps34 (1:500; Abcam, Cambrige, UK), anti-ATG14L (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-collagen (1:500;Novus Biologicals, CO, USA) and anti-fibronectin (1:500; abcam) antibodies. The cells were washed with blocking solution for 30 min and then incubated for 1 h with goat anti-rabbit Alexa Fluor® 594 (IgG) secondary antibody (Life Technologies, CA, USA) diluted in blocking solution. Cells were washed with PBS and then mounted with ProLongTM Gold Antifade Mountant with DAPI (Invitrogen). Cells were observed with the Nikon 80i microscope (Nikon, Tokyo, Japan) under the automatical exposure time for each wavelength or Zeiss LSM 880 with Airyscan confocal microscopy (Zeiss, Jena, Germany) wavelengths of 488 and 592 nm. Images were captured with the DP Controller software.
Anti atg14l
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Anti-Atg14L is a primary antibody that recognizes the Atg14L (Autophagy-related protein 14-like) protein. Atg14L is an important component of the PI3 kinase complex that is involved in the initiation of autophagy, a cellular process of degrading and recycling damaged or unwanted cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Anti beclin 1, by Santa Cruz Biotechnology (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Lsm 880, by Zeiss (1 mentions)
80i microscope, by Nikon (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Airyscan confocal microscopy, by Zeiss (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti hunk, by Thermo Fisher Scientific (1 mentions)
Anti rubicon, by Cell Signaling Technology (1 mentions)
Anti phosphoserine threonine, by Abcam (1 mentions)
Anti pi3k class 3, by Cell Signaling Technology (1 mentions)
Anti phosphoserine, by Abcam (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Anti gst, by Santa Cruz Biotechnology (1 mentions)
Anti myc, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
3 protocols using anti atg14l
1
Immunofluorescence Imaging of Autophagy Markers
2
Comprehensive Western Blot Analysis of Autophagy Regulators
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All cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100 with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). Primary antibodies used for Western blotting include: anti-Flag-M2 (Sigma-Aldrich, St. Louis, MO, USA, #F1804), anti-HUNK (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA, PA5-28765), anti-HA (Cell Signaling, Danvers, MA, USA, #3724), anti-GFP (Santa Cruz, CA, USA, #sc-8334), anti-LC3B (Cell Signaling, #2775), anti-Rubicon (Cell Signaling, #D9F7), anti-phosphoserine (Abcam, Cambridge, UK #9332), anti-phosphoserine/threonine (Abcam, #17464), anti-HA (Cell Signaling, #C29F4), anti-GST (Santa Cruz, sc-53909), anti-Beclin-1 (Santa Cruz, sc-48381), anti-UVRAG (ThermoFisher, #23215), anti-Atg14L (Cell Signaling, #5504), anti-PI3K class III (aka Vps34, Cell Signaling, #4263), and anti-β-tubulin (Santa Cruz, sc-55529). Imaging was performed on the FluorChem-R imaging system and quantitated using Alpha View SA software (Protein Simple, San Jose, CA, USA, version 3.4.0).
3
Western Blot Antibody Panel for Autophagy
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The commercial antibodies used for Western blot analysis include the following: Anti-phospho-Akt (Ser473) (1:1000 dilution; no. 3787), anti-LC3B (1:1000 dilution; no. 2775), anti-USP14 (rabbit, 1:1000 dilution; no. 11931), anti-Atg14L (1:1000 dilution; no. 5504), and anti-Flag (1:1000 dilution; no. 2368) were from Cell Signaling Technology. Anti-Beclin 1 (1:1000 dilution for Western blot [sc-11427]; 1:200 dilution for immunoprecipitation [sc-48341]), anti-Akt (1:1000 dilution; sc-8312), and anti-USP14 (mouse, 1:100 dilution for immunoprecipitation; sc-393872) were from Santa Cruz Biotechnology. Anti-Myc (1:1000 dilution; 16286-1-AP), anti-HA (1:1000 dilution; 51064-2-AP) anti-Rubion (1:1000 dilution; 21444-1-AP), and anti-GAPDH (1:10,000 dilution; 60004-1-Ig) were from Proteintech. Anti-Tubulin (1:10,000 dilution; PM054), anti-UVRAG (1:1000 dilution; PD027), and anti-p62 (1:5000; PM045) were from MBL. Anti-ubiquitin Lys63-specific antibody (1:2000 dilution; no. 2210353) was from Milllipore. Mouse monoclonal anti-Flag (1:500 for immunoprecipitation; F1804), Anti-Myc, and anti-HA affinity gel (E6779) were from Sigma-Aldrich. IU1 (S7134), MK2206 (S1078), E64D (S7379), rapamycin (S1039), and MG132 (S2619) were from Selleckchem.
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