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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Determining Internalization Rate of Cell-surface Proteins in Astrocytes by Biotinylation
NOTE: In the following, we describe a typical pulse-chase biotinylation experiment used in this instance to track the endocytosis of AQP4 in astrocytes. Specialized materials required include sulfo-NHS-SS-biotin, streptavidin-agarose resin, reduced glutathione, and iodoacetamide (see Table of Materials).
- Prepare cultures of mouse cortical astrocytes in 60-mm dishes using the methods outlined in the previous section. Ensure that cells are approximately 70% confluent on the day of the assay and that each dish contains an equivalent number of cells.
- Immediately prior to assay, prepare the following, and place on ice or refrigerate: CM-PBS or Phosphate buffered saline (100 mg/L MgCl2∙6H2O and 100 mg/L CaCl2 in 1X PBS, pH7.4), biotin buffer (0.5 mg/mL sulfo-NHS-SS-biotin in CM-PBS), reducing buffer (50 mM reduced glutathione, 75 mM NaCl and 75 mM NaOH), quenching buffer (50 mM iodoacetamide, 1% BSA, in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM EDTA or Ethylenediaminetetraacetic acid, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS or Sodium dodecyl sulfate, 30% glycerol, 300 mM DTT or Dithiothreitol and 0.01% bromophenol blue), and wash buffer (10 mM Tris, pH 7.4, 1.5 mM EDTA, 150 mM NaCl, 1% triton X-100, 1X protease inhibitor cocktail).
- Prepare fresh cell culture medium (Dulbecco's Modified Eagle Medium; or DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine), and place it in a 37 °C water bath.
- Remove astrocyte cultures from the incubator, and aspirate the medium using an aspirator.
- Wash cells thrice with 4 mL chilled CM-PBS, and place the dishes on crushed ice.
- Pipette 3 mL biotin buffer into each dish, tilt dishes back and forth a few times to ensure that the buffer is well-distributed, and leave on ice for 30 minutes.
- Aspirate biotin buffer, and replace it with 5 mL warm medium. Incubate a culture dish at 37 °C for 15 min, and a second dish at the same temperature for 30 min. Leave another dish at 4 °C as the 0 min sample.
- At the end of the incubation period, discard the medium and wash the cells thrice with 4 mL chilled CM-PBS. Pipette 6 mL reducing buffer over the cells and leave them on ice for 15 min.
- Remove the reducing buffer and replace it with 6 mL of fresh reducing buffer. Place the mixture on ice for an additional 15 minutes.
- Remove the reducing solution and replace it with 6 mL quenching buffer. Leave on ice for 15 min.
- Repeat the quenching step once more.
- Discard quenching buffer, and wash cells thrice with 4 mL chilled PBS.
- Using a cell lifter, scrape cells into 1 mL chilled PBS and transfer the suspension into a microcentrifuge tube.
- Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant, and resuspend cells in 500 µL lysis buffer.
- Leave this on ice for 30 min and vortex every 5 min. Alternatively, place the samples on an end-over-end rotator at 4 °C for this duration.
- Centrifuge the lysate at 14,000 x g for 10 min at 4 °C to pellet the detergent-insoluble materials, then transfer the supernatant to a new microcentrifuge tube. Save 50 µL of this lysate, add loading buffer to it, and denature at 95 °C in a dry bath; this is the "input" fraction, containing both biotinylated endocytosed proteins and nonbiotinylated proteins.
- Using a cut pipette tip, add 150 µL of the streptavidin-agarose slurry to the lysate, and incubate at 4 °C for 3 h on a shaker/rocker.
- Pellet streptavidin-agarose beads by centrifugation at 1,500 x g for 30 s at 4 °C.
- Resuspend beads in 1 mL wash buffer and rock for 3 min at 4 °C. Pellet beads (as per step 1.18) and discard the supernatant. Repeat this process 4 times to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.
- Pellet beads by centrifugation at 1,500 x g for 30 s at 4 °C and discard the overlying wash buffer. Add 50 µL 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing at 95 °C; this fraction should contain internalized cell-surface proteins only ("endocytosed" fraction).
- Separate input, cell-surface, and unbound fractions by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by western blotting.
NOTE: While we used a 4 - 20% precast gradient gel in our experiments, a 12 - 14% separating gel with a 4% stacking layer (each containing 0.1% SDS) should suffice for the proteins of interest in this study. A molecular weight standard of the appropriate size range should also be used. Note that there can sometimes be an observable upshift in the apparent molecular masses of biotinylated proteins.