Confocal microscopy was used to determine M. tuberculosis–induced autophagic flux and colocalization of M. tuberculosis with the autophagosome marker LC3B and the autophagy receptor p62 in J774.1 macrophages. Briefly, the cells were allowed to adhere to sterile glass coverslips placed in 6-well tissue culture plates, prestimulated with IFN-γ, as described above, and infected with an M. tuberculosis CDC1551 strain ectopically expressing GFP at an MOI of 10 for 6 hours. Cells were fixed, permeabilized, and immunostained using either anti-LC3 antibody (Novus NB100-2220) or anti-p62 antibody (Sigma-Aldrich P0067). Cells were then washed and incubated with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific). Hoechst 33342 was used for nuclear staining. Image acquisition was carried out using an LSM700 confocal microscope at ×63 original magnification. Image processing and analysis were done using Fiji (https://github.com/fiji/fiji ) and Imaris 9.7 software (Oxford Instruments). For LC3 and p62 quantification, perinuclear puncta were counted in a minimum of 200 cells in different fields using Imaris 9.7 and GraphPad Prism software.
P0067
Manufactured by Merck Group
Sourced in United States
P0067 is a laboratory equipment product. It serves as a core function in the laboratory setting. Detailed specification and intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
L7543, by Merck Group (4 mentions)
T5326, by Merck Group (3 mentions)
Imaris 9, by GraphPad (2 mentions)
Nb100 2220, by Merck Group (2 mentions)
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Imaris 9, by Oxford Instruments (2 mentions)
Nb100 2220, by Novus Biologicals (2 mentions)
Sc 9996, by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
L7543, by Abcam (2 mentions)
Ab137029, by Proteintech (2 mentions)
Sc 271595, by Proteintech (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Ab192890, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab41407, by Abcam (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Ab6046, by Abcam (1 mentions)
Phospho gsk3β, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
31 protocols using p0067
1
Quantifying Mycobacterium tuberculosis Autophagy in Macrophages
2
Antibody Validation and Reagent Use
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Antibodies against the following proteins were used in this study: p62 (P0067, Sigma-Aldrich), LC3b (L7543, Sigma), GAPDH (60004-1-Ig, proteitech), FLAG (F3165, Sigma-Aldrich), GFP (AB513, Evrogen), p53 (ET1601–13, HuaAn Biotechnolog), phospho-Histone H3 (ab11477, abcam), phospho-Akt (#4060S, Cell Signaling), Akt (#9272, Cell Signaling), phospho-GSK-3β (#9323, Cell Signaling), GSK-3β (#9315, Cell Signaling), β-tubulin (ab6046, Abcam), HDAC1 (ab41407, Abcam) and zebrafish CD44a (25340-1hz, Abmart). Secondary antibodies for immunoblotting or immunofluorescence used in this study: goat anti-rabbit IgG and goat anti-mouse IgG (Prod #31460 and #31430, pierce), ReadyProbes™ Alexa Fluor® 594 Goat Anti-Mouse IgG Antibody (#R37121, Invitrogen), Alexa Fluor™ 488 Goat anti-Rabbit IgG (#A11008, Invitrogen) and Alexa Fluor™ 488 (#A110011, Invitrogen) Goat anti-Mouse IgG. The chemicals used in this study: Z-IETD-FMK (50 μm; #S7314, Selleck), pifithrin-μ (50 μm; #S2930, Selleck), pifithrin-α (5 μm; #S2929, Selleck), rapamycin (200 nm; #S1039, Selleck) and CQ (100 μm; #C6628, Sigma-Aldrich) and MitoTracker Deep red (Invitrogen™, M22426).
3
Western Blot Analysis of Autophagy Markers
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Proteins of brain homogenates (20 μg protein) were denatured using 5x Laemmli sample buffer (25% (v/v) 1.25 M Tris-HCL pH 6.8, 50% (v/v) 100% glycerol, 10% (w/v) sodium dodecyl sulfate, 8% (w/v) DTT, and 0.1% (w/v) bromophenol blue), samples were boiled for 5 min at 98°C, and proteins were separated by electrophoresis on a 12% (w/v) SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes (0.2 μM, Bio-Rad laboratories Inc., Hercules, USA) using the Trans-Blot® Turbo™ Transfer system (Bio-Rad). Membranes were blocked with 5% (w/v) BSA and incubated overnight at 4°C with primary antibodies: rabbit anti-LC3 (1:1000, NB100-2220; Novus Biologicals, Centennial, USA), rabbit anti-p62/SQSTM1 (1:1000, P0067; Sigma-Aldrich), or rabbit anti-actin (1:1000, ab209857; Abcam, Cambridge, UK). Membranes were washed 3 times with TBST and incubated for 1 h at RT with secondary antibody: GARPO goat anti rabbit IgG (H+L) peroxidase (1:5000, Bio-Rad). Chemiluminescence signal was developed using the Clarity Max Western ECL substrate (Bio-Rad), detected using a ChemiDocMP imager (Bio-Rad) and the signal quantified by ImageJ software.
4
Quantifying Autophagy in HeLa Cells
5
Antibody Selection and Characterization
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Antibodies and their manufacturers were as follows: anti-β-actin (AP0060; Bioworld Technology, St. Louis Park, MN, USA), anti-α glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000) (AT0002; CMCTAG, Dover, DE, USA), anti-GFP (1:5000) (AT0028; CMCTAG), anti-FLAG (1:5000) (AT0022; CMCTAG), anti-LC3 (1:3000) (L7543; Sigma-Aldrich), anti-p62 (1:5000) (P0067; Sigma-Aldrich), and anti-14-3-3ε (1:800) (sc-23957; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-NSP2 antibodies were prepared by immunizing New Zealand white rabbits with a recombinant protein composed of the N-terminal 180 amino acids of NSP2 (NSP2-180). The monoclonal antibody against PRRSV-2 nucleocapsid (N) protein 6D10 was prepared in our laboratory [44 (link)]. Horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were purchased from Jackson Laboratories (1:5000) (West Grove, PA, USA).
Complete™ protease inhibitor cocktail (04693132001) was purchased from Roche (Basel, Switzerland), the aggresome detection kit (ab139486) was purchased from Abcam (Cambridge, UK), and nocodazole (M1404) and MG132 (C2211) were obtained from Sigma-Aldrich.
Complete™ protease inhibitor cocktail (04693132001) was purchased from Roche (Basel, Switzerland), the aggresome detection kit (ab139486) was purchased from Abcam (Cambridge, UK), and nocodazole (M1404) and MG132 (C2211) were obtained from Sigma-Aldrich.
6
Autophagy Evaluation Protocol
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7
Immunostaining of p62 in Murine Fibroblasts
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Murine embryonic fibroblast cells grown in a glass‐bottom dish were fixed with 4% PBS for 15 min at room temperature. After washing with 0.1% Tween 20 in PBS (PBST), cells were permeabilized with 0.5% Triton X‐100 in PBS for 5 min at room temperature and washed with PBST three times. The cells were then blocked with Blocking One solution (03953‐95; Nacalai Tesque) for 30 min at 4 °C, washed once with PBST, and incubated with a rabbit polyclonal anti‐p62 antibody (P0067; Sigma‐Aldrich) in Can Get Signal Immunostain Solution A (NKB‐501; Toyobo, Osaka, Japan) for 2 h, followed by extensive washes and incubation with Cy3‐conjugated donkey anti‐rabbit IgG antibody (AP182C; Life Technologies, Carlsbad, CA, USA) for 1 h. After washing with PBST three times, cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and subjected to FM. For observation, an oil‐immersion objective lens (PLAPON60xOSC/NA1.40; Olympus) on the DeltaVision microscope system (GE Healthcare Life Sciences) was used as previously described 25 .
8
Autophagy and Neural Stem Cell Assays
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Baf.A1 (BML-CM110, Enzo, USA), pepstatin A (PepA, P5318, Sigma-Aldrich, USA), E64d (E8640, Sigma-Aldrich), puromycin (NC9138068, Invitrogen, USA), hygromycin B (H0192, Duchefa, The Netherlands), fetal bovine serum (FBS, #101, Tissue Culture Biologicals, USA), forskolin (BML-CN100, Enzo), and retinoic acid (RA, #BML-GR100, Enzo) were purchased from the indicated companies. Horseradish peroxidase-conjugated β-actin (SC-47778, Santa Cruz Biotechnology, USA) and antibodies against ATG7 (#8558, Cell Signaling Technology, USA), LC3 (NB100-2220, Novus Biologicals, USA), p62 (P0067, Sigma-Aldrich, USA), sex-determining region Y-box 2 (Sox2, ab97959, Abcam, UK), microtubule-associated protein 2 (MAP2, ab5392, Abcam), receptor interaction protein (RIP, MAB1580, Merck, USA), glial fibrillary acidic protein (GFAP, NBP1-05198, Novus Biologicals), nestin (bs-0008R-A555, Bioss, USA), and GFP (SC-9996, Santa Cruz Biotechnology) were purchased from the indicated companies.
9
Immunofluorescence analysis of autophagy markers
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The extracted eyeballs or mouse corneal stromal cells were fixed in 4% paraformaldehyde. Next, samples were treated with 0.1% Triton X-100 for 5 minutes and blocked using 5% BSA for 1 hour. Afterward, incubated with rabbit anti-p62 (P0067; Sigma) and rabbit anti-ATG5 (A0856; Sigma) at 4°C overnight, and the sections were washed and incubated with Alexa Fluor 488 goat anti-rabbit (ab150077; Abcam, Cambridge, UK) or Alexa Fluor 594 goat anti-rabbit (ab150080; Abcam) secondary antibodies in the dark for 1 hour at 37°C. Isotype IgG was used as the negative control and nuclei were visualized with DAPI (Beyotime Biotechnology, Shanghai, China). Images were captured with a fluorescence microscope.
10
Protein Expression Analysis Protocol
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Analysis of p62/SQSTM1, LC3 (P0067 and L7543, Sigma-Aldrich), ERα, GFP (sc-8005 and sc-9996, Santa Cruz Biotechnology), pAkt, and Akt (9271S and 4691, respectively, Cell Signaling, Danvers, MA, USA), and normalization versus β-actin (A5441, Sigma-Aldrich) were performed as described [21 (link)]. Bands were detected with the ChemiDoc XRS+ Imaging System (Bio-Rad Laboratories).
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