Phosphate deprivation prompted a tissue-specific alteration in methylome patterns, encompassing phosphate assimilation genes and, unexpectedly, sulfate metabolism genes not previously associated with this phenomenon. MethylIT's utility in identifying stage-specific gene networks was demonstrated through its ...
After centrifugation, adipocytes in the supernatant and SVF in the pellet were collected and washed with phosphate buffer saline (PBS) twice. Immunoblot Adipose tissue, SVF or adipocytes were lysed with lysis buffer (50 mM Tris-HCl, 1% SDS, pH 7.4) supplemented with ethylene diamine ...
Cells were broken with glass beads in lysis buffer (50 mM sodium phosphate, 150 mM Nacl, 2 mM EDTA, 0.1% (v/v) NP-40, pH 7.4; plus protease inhibitors) using a Minibead beater (Biospec Scientific, Bartlesville) for 45 s at 4 °C The supernatant was centrifuged at 13,000g for 5...
The pellet containing the mitochondria-enriched fraction was resuspended in cold supplemented RIPA Buffer 1x (NaCl 150 mM; Tris-HCl 50 mM; NP-40 1%; sodium deoxycholate 0.5%; SDS 0.1%). The supernatant was enriched in the microsomal fraction. Western blotting The cells were lysed in ...
In control reactions, phosphatase inhibitors (10 mM NaF, 10 mM p-nitrophenylphosphate, 10 mM sodiumpyrophosphate, and 10 mM β-glycerophosphate) were added. Miscellaneous Northern analyses, trehalose measurements, flow cytometry, and indirect immunofluorescence were performed as described Reinders et al....
The mpkCCD cells were washed twice with phosphate-buffered saline (PBS) and then solubilized in 200 µL of lysis buffer as previously described32. For analysis of the cell fractions, the mpkCCD cells were incubated with 650 µL of 1% Triton X-100 in PBS for 3 min before cell...
Calcium phosphate transfections HEK cells were seeded onto 60 mm dishes at ~ 70% den- sity 24 h prior to transfection. Transfection media was prepared by resuspending DNA (10–25 µg) in TE Buffer (220 µl total volume); 30 µL of 2 M CaCl2 was then ...
[30,31,32]. To gain insight into the regulatory mechanism of ChA biosynthesis inC. globosum, in the previous study, we knocked down the glucosamine 6-phosphateN-acetyltransferase 1 (gna1) gene (homolog of CHGG_03321 ofC. globosumCBS 148.51), which putatively encodes a group I Gα protein,...
HEK293T cells were transfected and lysed in cell lysis buffer (10 mm sodium phosphate buffer (pH 6.95), 150 mm NaCl, 5 mm EDTA, 5 mm EGTA, and 1% Triton X-100) as described previously (24). For phosphorylation experiments, the phosphatase inhibitor microcystin-LR was included (100 nm)...
Phosphate-Buffered Saline (PBS) 28.5 g of Na2HPO4×2H2O 5.5 g of NaH2PO4×H2O 164 g of NaCl ad 1 l H2O Mix of Protease Inhibitors 1.4 μg/μl trasylol (aprotinin) 0.5 mM benzamidine 3.2 μg/μl soy bean trypsin inhibitor (STI) Phenylmethylsulfonyl fluoride (PMSF) Solution (40 mM...