The aim of this presentation is to show how western blotting and immunocytochemistry can be used to demonstrate the SorLA and CLC:CLF-1 mediated downregulation and lysosomal degradation of the CNTF receptor in human cell transfectants. This method can help answer key questions regarding the turnover of the GPI-anchored CNTF receptor upon its co-expression with the endocytic receptor SorLA and exposure to CLC:CLF which interconnects the two. The main advantage of this technique is that it does not involve radioactivity and is suited for membrane proteins which exhibit only a small degree of degradation and use synthesis in unstimulated cells.
Use stable clones of human embryonic kidney 293 transfectants expressing Myc-tagged CNTF receptor or co-expressing Myc-tagged CNTF receptor and SorLA. Seed equal numbers of cells in two four-well plates. Culture the cells in full medium which is DMEM supplemented with 10%FBS, penicillin, and streptomycin in a 5%carbon dioxide incubator at 37 degrees Celsius.
Once the cells are 50 to 80%confluent, remove the medium from the wells. After washing once with Dulbecco PBS, add fresh full medium with or without 10 nanomolar CLC:CLF-1 to each cell type on both plates. Then incubate the cells at 37 degrees Celsius.
After zero and five hours of incubation, recover the medium and store it at 4 degree Celsius in 1.5 milliliter tubes. Then add 1%Triton X-100 lysis buffer supplemented with a protease inhibitor cocktail to each well. Subsequently transfer the cell lysates to the 1.5 milliliter tubes.
Next, spin the tubes containing the medium and cell lysate. When finished, transfer the supernatants to 1.5 milliliter tubes and discard the pellets. Transfer five microliters of each cell lysate supernatant to a 1.5 milliliter tube to be used for measuring the protein content.
Then immediately add non-reducing LDS sample buffer to all the supernatants and vortex the samples. Measure the protein content in the cell lysate supernatants using a commercial protein assay according to the manufacturer's protocol. Following this, heat the samples at 95 degrees Celsius for five minutes.
Subject an equal amount of protein from each sample to reducing SDS page and Western blotting and visualize the content of SorLA, beta-actin, and Myc-tagged CNTF receptor in the medium and in the cell lysates. Quantify the individual bands by densitometry according to the manufacturer's protocol. Seed human embryonic kidney 293 cells stably transfected with Myc-tagged CNTF receptor and SorLA on cover slides in a four-well plate.
Allow the cells to attach to the cover slides by incubating them at 37 degrees Celsius for one hour. Culture the cells in full medium as previously described. Once the cells are 20 to 40%confluent, remove the medium from the wells and add full medium with or without 50 micrograms per milliliter of leupeptin and of pepstatin A to the cells.
Then incubate the cells at 37 degrees Celsius. The leupeptin and pepstatin A medium must be replaced approximately every six hours for 24 hours. Failure to do so may result in active lysosomal enzymes and less or no detectable accumulation of proteins in the lysosomes.
Following 24 hours of incubation, replace the medium and add 10 nanomolar of CLC:CLF-1. After incubating the cells at 37 degrees Celsius for five hours, wash them once in PBS and fix them in 4%paraformaldehyde for 15 minutes at room temperature. Once the cells have been washed once in PBS, permeabilize them for five minutes at room temperature in 0.5%saponin in PBS.
Next, dilute goat anti-CNTF receptor and mouse anti-LAMP-1 antibodies in 0.5%saponin in PBS and incubate the cells with the antibodies for two hours at room temperature. After washing the cells four times in 0.5%saponin in PBS, incubate them with six micrograms per milliliter each of donkey anti-goat Alexa 488 and donkey anti-mouse Alexa 568 conjugated secondary antibodies and incubate for two hours at room temperature in the dark. Following this, wash the cells four times in 0.5%saponin in PBS and two times in deionized water.
Then mount the cover slides on microscope slides using mounting media. Analyze the antibody staining of the cells by confocal microscopy using a laser scanning confocal microscope. Seed human embryonic kidney SorLA cells in a four-well plate.
Culture the cells in full medium as previously described. Once the cells are 50 to 80%confluent, remove the medium from the wells. Wash once in PBS and add fresh full medium with or without 10 nanomolar of CLC:CLF-1 to the cells.
After incubating the cells at 37 degrees Celsius for five hours, remove the medium. Then wash the cells twice in blank medium which is unsupplemented DMEM. Following re-incubation for 90 minutes, remove the medium and add fresh blank medium with or without five nanomolar of CLC:CLF-1.
Then incubate for 15 minutes at 37 degrees Celsius. Following this, quickly remove the medium and lyse the cells by adding 1%Triton X-100 lysis buffer supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail to each well. Subsequently, transfer the cell lysates to 1.5 milliliter tubes.
Next, transfer five microliters of each supernatant to a 1.5 milliliter tube to be used to measure the protein content. Then immediately add non-reducing LDS sample buffer to all the supernatants and vortex the samples. Immediate addition of sample buffer is important to stop the reaction and to prevent dephosphorylation.
Measure the protein content of the cell lysate supernatants using a commercial protein assay according to the manufacturer's protocol. Now sonicate each sample at room temperature until they are no longer viscous. Subsequently heat the samples at 95 degrees Celsius for five minutes.
Subject an equal amount of protein from each sample to SDS page and western blotting and visualize the content of phosphorylated STAT3 and total STAT3 in the cell lysates using the appropriate anti-STAT3 antibodies and HRP-linked secondary antibodies. The SorLA-mediated CLC:CLF-1-dependent downregulation of CNTF receptor is shown here. Note that the bands representing Myc-tagged CNTF receptor and CLC:CLF-1 stimulated and unstimulated single transfectants are similar at both time points.
In contrast, downregulation of Myc-tagged CNTF receptor is reflected by the comparatively weak band in stimulated double transfectants after five hours. The SorLA-mediated endocytosis and lysosomal targeting of CNTF receptor shows that Myc-tagged CNTF receptor has accumulated in LAMP-1 positive vesicles of leupeptin and pepstatin A treated cells. In contrast, no accumulation of Myc-tagged CNTF receptor is seen in cells not subjected to treatment which demonstrates that Myc-tagged CNTF receptor is sorted to lysosomes and degraded.
The level of pSTAT3 is significantly reduced in pre-stimulated cells as compared to the level in cells that have not been pre-exposed to CLC:CLF-1. This is in accordance with the observed SorLA-mediated downregulation of CNTF receptor in the pre-stimulated cells as shown previously. After watching this video, you should have a good understanding of how to use Western blotting and immunocytochemistry in order to show the SorLA-mediated and CLC:CLF-1-dependent downregulation and lysosomal degradation of the CNTF receptor.
