1/9/2024 0 Comments Noti enzymeThe cDNA for PI4K2B was originally from Addgene (pDONR223-PI4K2B, 23507) which contained a 15-bp insert in the middle that introduced a premature stop codon, leading to the production of an inactive fragment majorly localized into the nucleus. The ORP11 cDNA was purchased from Sino Biological (HG22085-U). All cDNA sequences were of human origin unless otherwise specified. DNA cloningįor stable expression of proteins in this study, the relevant DNA sequences were cloned into pCDH-CMV-MCS between restriction sites BamHI and NotI. Alexa-488/594- and Pacific Blue-conjugated secondary antibodies were obtained from ThermoFisher Scientific. The following antibodies were from Bethyl Laboratories: ORP11 (A304-580A, western blot 1:2,000, immunofluorescence 1:1,000) and ORP10 (A304-885A, western blot 1:1,000). Mouse anti-Alix antibody (634502, immunofluorescence 1:1,000) was from Biolegend. Rabbit anti-LAMP1 monoclonal antibody (9091, immunofluorescence 1:100) was from Cell Signaling. Anti-rabbit IgG (H+L) CF350 (SAB4600412, immunofluorescence 1:200), Flag (M2, immunofluorescence 1:1,000 F7425, western blot 1:3,000), Flag M2 agarose (immunoprecipitation 10 μl beads per reaction) were from Sigma. The PI4KB antibody (611816, western blot 1:1,000, immunofluorescence 1:200) and GM130 antibody (610822, immunofluorescence 1:1,000) were from BD Biosciences. For cell treatment that requires medium change, fresh media were pre-warmed overnight in an empty dish in the same incubator, which minimizes disturbance to cells caused by medium change. Chemicals, silica, or tau fibrils were directly added to cell culture media to induce lysosomal membrane damage with equal volume of vehicle (ethanol or DMSO) added into control wells. Tau fibrils (SPR-329) were from StressMarq. Nano-silica (Invivogen, tlrl-sio-2) was suspended in water and sonicated right before each use. Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, 14634), Brefeldin A (Sigma, B5936), ML-SA1 (Cayman Chemical, 29958), MEK6–83 (Cayman Chemical, 21944), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, 850546) were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 ☌. LLOME (Sigma, L7393) was dissolved in ethanol and stored in aliquots at −20 ☌. All cells were maintained at 37 ☌ with 5% CO 2. Both media were supplemented with 8% fetal bovine serum (FBS) and penicillin/streptomycin. 293T, U2OS, COS7, and BJ cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) PC3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640. All cell lines used in this study were free from mycoplasma contamination based on PCR detection and were regularly maintained with mycoplasma reagent. Cell lines in this study have different morphologies and growth rates and contamination were constantly monitored. 293T, U2OS, PC3 and BJ cells were authenticated through short tandem repeat profiling and the profiling data are publicly available from ATCC.
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