Microfuge 22r microcentrifuge

Cell lysate collected from C6/36 cells transfected with the plasmid pAC5.1-HA-C189 was centrifuged using a Microfuge 22R microcentrifuge (Beckman Coulter), and PNS was harvested for further magnetic immunoisolation by using a Dynabeads Protein G Immunoprecipitation Kit (Invitrogen). To prepare anti-HA Dynabeads, we added 50 μl of them coated with protein G into the 8-tube Magnetic Separation Rack to remove the supernatant. Then, 100 μl antibody binding and washing buffer containing 10 μg mouse anti-HA IgG (Sigma) was added into the tube and gently shaken for 1 h. After another wash with antibody binding and washing buffer and centrifugation to remove the supernatant, prepared anti-HA Dynabeads were collected and subsequently mixed with PNS. After shaking at RT for 1 h, the mixture was transferred to an 8-tube Magnetic Separation Rack and washed three times with 200 μl of washing buffer, followed by adding100 μl of fresh washing buffer to resuspend the mixture and then moved into a new collection tube. For protein analysis, a mixture of 30 μl 1x Laemmli sample buffer was heated in a water bath at 95°C for 5 min. Protein was then collected from the supernatant from the 8-tube Magnetic Separation Rack and subjected to Western blot analysis. For RNA detection, samples were RNA-extracted via a GENEzol TriRNA Pure Kit (Geneaid Biotech).

In order to collect the cell lysate from C6/36 cells transfected with the plasmid pAC5.1-HA-C189, transfected cells were washed with PBS. Cells were scraped from the dish after adding 10% sucrose with lysis buffer (pH 7.4) containing 3 mM imidazole, 1 mM EDTA, RNase inhibitor (Sigma, St. Louis, MO, USA), and protease inhibitor cocktail (AG Scientific, San Diego, CA, USA). The cell lysate was then collected by disrupting cells using a 1 ml syringe with 27 G needle for at least 30 strokes, followed by centrifugation using a Microfuge® 22R microcentrifuge with F241.5P Rotor (Beckman Coulter, Atlanta, GA, USA) at 3500 rpm for 10 min. Post nuclear supernatant (PNS) collected after centrifugation was added to the top of a column containing 10-60% sucrose gradients containing 3 mM imidazole and 1 mM EDTA, from top to bottom. The column was then centrifuged in an Optima L-90K Ultracentrifuge with SW-41Ti Rotor (Beckman Coulter) at 41000 rpm at 4°C for 18 h. Subsequently, fractions containing 0.5 μl were harvested from the top to bottom, with up to 23 fractions being collected for further analysis for RNA and protein.

RNA extraction was carried out with a GENEzol TriRNA Pure Kit (Geneaid Biotech), following manufacturer instructions. In brief, 300 μl samples collected from sucrose gradient centrifugation were mixed with 300 μl GENEzol Reagent. After incubation for 5 min, 600 μl of absolute ethanol was added. The mixture was subsequently transferred to an RB column set with a 2 ml collection tube, which was then centrifuged in a Microfuge 22R microcentrifuge (Beckman Coulter) at 14000 g for 1 min. Subsequently, a new 2 ml collection tube was substituted and 400 μl of buffer added for a prewash and then centrifuged at 14000 g for 1 min. Another two washes were done with 600 μl wash buffer and then centrifuged at 14000 g for 1 min, followed by the last 3 min centrifugation wash under the same conditions. A clean 1.5 ml microcentrifuge tube was then set up into the RB column in which 30 μl DEPC-ddH₂O was added. After 5 min of incubation, the column was centrifuged for 3 min at 14000 g. All collected samples were referred to RT-PCR analysis. For the magnetic immunoisolation samples, 5 × 10⁶ cells were added and mixed with 700 μl GENEzol Reagent (Geneaid), but otherwise treated the same as those mentioned above.