The following primary antibodies were used: rabbit polyclonal anti-VMP1 (ab240887; Abcam; UK); mouse monoclonal anti-flag (AE005; Abclonal; China); rabbit polyclonal anti-Na + -K + -ATPase (ab76020; Abcam); rabbit polyclonal anti-EEA1 (ab2900; Abcam); rabbit monoclonal anti-Rab7 (ab137029; Abcam); rabbit polyclonal anti-Rab11 (#71–5300; invitrogen; USA); mouse monoclonal anti-CD63 (ab217345; Abcam; UK); rabbit polyclonal anti-Alix (12422–1-AP; Proteintech; China); rabbit polyclonal anti-Tsg101 (ab83; Abcam); mouse monoclonal anti-LAMP1 (sc-19992; Santa cruz; USA); mouse monoclonal anti-CHMP4 (sc-514869; Santa cruz; USA). 2-BP (#21604) was purchased from Sigma Aldrich. Baf A1(#54645) was purchased from Cell Signaling Technology; When indicated, the medium contained 50 μM 2-BP or 20 nM Baf A1.
Baf a1
Manufactured by Cell Signaling Technology
Baf A1 is a specific inhibitor of vacuolar-type H+-ATPase (V-ATPase). It blocks the proton translocation activity of V-ATPase, which is responsible for acidifying intracellular compartments such as lysosomes and endosomes.
Automatically generated - may contain errors
Lab products found in correlation
Pngase f, by Merck Group (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Gw4869, by Merck Group (2 mentions)
Endo h, by New England Biolabs (2 mentions)
Mg132, by Merck Group (2 mentions)
Ab76020, by Abcam (1 mentions)
Ab2900, by Abcam (1 mentions)
Ab137029, by Abcam (1 mentions)
Ab217345, by Abcam (1 mentions)
Ae005, by ABclonal (1 mentions)
Sc 19992, by Santa Cruz Biotechnology (1 mentions)
Antimycotic, by Thermo Fisher Scientific (1 mentions)
Wortmannin, by Thermo Fisher Scientific (1 mentions)
Jte 013, by Cayman Chemical (1 mentions)
Easysep mouse neutrophil enrichment kit, by STEMCELL (1 mentions)
Ty 52156, by Cayman Chemical (1 mentions)
L5293, by Merck Group (1 mentions)
Bafilomycin a1, by Cell Signaling Technology (1 mentions)
G7940, by Promega (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
7 protocols using baf a1
1
Intracellular Trafficking Protein Analysis
2
Glycosylation and Trafficking Regulation of PD-L1
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To inhibit lysosome function, cells were cultured with 250 nM BafA1 (Cell Signaling, cat. 54645S) for six hours. To inhibit proteasome function, cells were cultured in 10 uM of MG132 (SIGMA, cat, M7449) for six hours.
To investigate PD-L1 glycosylation, PC3 cell lysate were treated with PNGase F (Sigma-Aldrich, cat. P7367). To investigate further glycosyl modification in the Golgi apparatus, PC3 cell lysate were treated with Endo H (NEB, cat. P0702S).
For IFN-γ treatments, 10ng/mL IFN-γ (PeproTech – human cells, cat. 300–02) (Abcam – mouse cells, cat. Ab9922) to media for 48 hours prior to exosome and cell collections.
For GW4869 treatments, 10μM GW4869 (Sigma-Aldrich, cat. D1692) to media for 48 hours prior media collections.
To investigate PD-L1 glycosylation, PC3 cell lysate were treated with PNGase F (Sigma-Aldrich, cat. P7367). To investigate further glycosyl modification in the Golgi apparatus, PC3 cell lysate were treated with Endo H (NEB, cat. P0702S).
For IFN-γ treatments, 10ng/mL IFN-γ (PeproTech – human cells, cat. 300–02) (Abcam – mouse cells, cat. Ab9922) to media for 48 hours prior to exosome and cell collections.
For GW4869 treatments, 10μM GW4869 (Sigma-Aldrich, cat. D1692) to media for 48 hours prior media collections.
3
Neutrophil Isolation and Stimulation Assay
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Bone marrow–derived neutrophils were isolated from femurs/tibias by using the EasySep Mouse Neutrophil Enrichment Kit (19762, Stem Cell Technologies). Freshly isolated neutrophils were cultured (1.5 × 106 cells/mL in RPMI supplemented with 2% FBS and Antimycotic; 15240062, Thermo Fisher) and stimulated with S1P (SML2709, Millipore Sigma) or LPS (L5293, Millipore Sigma). In some experiments, neutrophils were pretreated with S1PR2 antagonist (JTE-013, 10009458, Cayman Chemical), S1PR3 antagonist (TY 52156, 19119, Cayman Chemical), autophagy antagonists, wortmannin (12-325, Fisher Scientific) or Baf A1 (54645S, Cell Signaling Technology), or CathB inhibitor (CA-074, 4846, Bio-Techne Corp.).
4
Glycosylation and Trafficking Regulation of PD-L1
5
Titanium Modulates Autophagy in Osteoblasts
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The effect of Ti ions on autophagy in osteoblastic cells was evaluated. Cells were treated with TiCl4 (cat. no. 7550-45-0; Shanghai Aladdin Bio-Chem Technology Co., Ltd.) at different concentrations (0, 10, 25 and 50 µM) at 37°C for 24 h. The cells were also treated with various concentrations (0, 10, 25 and 50 µM) of TiCl4 for 24 h with or without lysosomal inhibitor bafilomycin A1 (Baf-A1; 20 nM; Cell Signaling Technology, Inc.) at 37°C for 24 h. The vehicle control used for each in vitro assay was 0.1% DMSO. The role of the sirtuin3 (SIRT3)/superoxide dismutase 2 (SOD2) pathway in osteoblasts was investigated.
6
ADE Inhibition Assay with Raji Cells
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The ADE inhibition assay were performed using Raji cells. Diluted mAbs (MW05: 750 ng/mL and MW05/Mu: 200 ng/mL) were mixed with pseudovirus. The mixture was incubated for 60 min at 37 °C, supplied with 5% CO2. 25 µL of serially diluted inhibitors of micropinocytosis and endocytosis. (Dynole, Abcam ab120346; methyl-β-cyclodextrin, Sigma C4555-1G, Thioridazine, Sigma T9025-5G; Apilimod, MCE HY-14644; Cytochalasin D, MCE HY6682; Fillipin, CAYMAM CHEMICAM COMPANY 70400; Baf.A1,Cell Signaling 56545 S) were mixed with 25 µL cells at the density of 4 × 106 cells/mL. Then add the mixtures of pseudoviruses and mAbs to the cell plate for an additional 24 h incubation. Then, the same volume of luciferase detecting regents (Promega G7940) was added to each well. After 2 min of incubation, the luciferase activity was measured using a microplate luminometer (SpectraMax i3x MolecularDevices). For evaluation whether FcγRI expression on B cells could impact the ADE mediated by MW05 or not, the construct FcγRI-P2A-gamma chain driven by SFFV promoter was generated. Raji cells were used for ADE measurement after transfected by FcγRI-P2A-gamma chain construct with electroporation strategy.
7
Mitochondrial Respiration Assay
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Acn (Enzo Life Sciences or Cayman Chemicals) was used at 200 µM, FCCP (Enzo Life Sciences) was used at 20 µM; BafA1 (Cell Signaling Technology) was used at 100 nM; CP (Sigma-Aldrich) was used at 200 µM; rotenone (Sigma-Aldrich) was used at 50 µM; the mixture of oligomycin (Sigma-Aldrich) and antimycin A (OA; Sigma-Aldrich) was used at 10 µg/ml and 5 µg/ml, respectively; ABT-737 (Axxora) was used at 10 µM; and TMRM (Thermo Fisher Scientific) was used at 50 nM.
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