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Escirt Less popularcategories such milf mature year also be e. Isoproterenol, leupeptin, monoclonal FLAGM1, polyclonal FLAG, rabbit anti-actin, alkaline phosphatase-conjugated goat anti-mouse antibodies, and the alkaline phosphatase substrate kit were purchased from Sigma-Aldrich. Deletion and site-directed mutagenesis were carried out by PCR. Integrity sherbroome the coding sequence of these constructs was confirmed by dideoxy sequencing. Immunoprecipitations HEK cells were transiently transfected with the indicated constructs and were maintained as described above for 48 h.
One microgram of specific antibodies was added to the supernatant. Samples were then centrifuged for 1 min in a microcentrifuge and washed three times with 1 ml of lysis buffer. Subsequently, the cells were washed three times with PBS and permeabilized by incubation for 10 min with 0. After two washing steps with 0. The cells were washed twice with the permeabilization buffer, blocked again for 10 min, and incubated with the appropriate secondary antibody for 60 min at room temperature. Deglycosylation Assays Receptors transiently expressed in HEK cells in mm plates were immunoprecipitated as above. Samples were washed three times with lysis buffer.
For PNGase F experiments, immunoprecipitated proteins were eluted by denaturation with 0. Cells were transfected with the indicated constructs and then maintained for an additional 48 h.
The cells were fixed in 3. Cells Esvort then incubated with an alkaline phosphatase-conjugated goat anti-mouse antibody at 1: The recombinant proteins shebrrooke purified using nickel-nitrilotriacetic acid-agarose resin Qiagen as indicated by the manufacturer. The binding reactions were then washed three times with binding buffer. SDS sample buffer was added to the binding reactions, and the tubes were boiled for 5 min. The pulldown reactions were analyzed by Western blotting with the indicated specific antibodies. HEK cells were transfected with 20 nm oligonucleotide using the Lipofectamine transfection reagent Invitrogen according to the manufacturer's indications except for the following modifications.
Cells were seeded directly into the transfection mix at twice the cell density as indicated in the basic protocol. Statistical Analysis Statistical analysis was performed using Prism version 5. A higher molecular mass form of Rab11a was detected after longer exposure of Western blot membranes, and this form was increased when GPCRs were co-expressed in HEK cells.