PART A: ErbB-2 is a receptor found on the plasma membrane of mammalian cells. Recent studies have shown that this protein can move from the plasma membrane to the nucleus. In the first experiment, RNA interference (siRNA) is used to reduce levels of importin \(\beta\)1. Cells are either not subjected to siRNA (-), or have their expression of importin \(\beta\)1 reduced (imp), or are subjected to a nonsense siRNA (NS). The presence of ErbB-2 in both cytoplasmic and nuclear fractions is then analyzed by immunoblotting using antibodies against each of these proteins. The data are shown on the immunoblot below in Figure (a). In the second experiment, lysates of cells expressing either wild-type (WT) ErbB-2 or mutant ErbB-2 lacking its nuclear localization signal (\(\Delta\)NLS) are immunoprecipitated with antibody to ErbB-2 or a control antibody (mIgG). The immune-precipitates are then examined by immunoblotting with antibodies to importin \(\beta\)1 or to ErbB-2 as shown in Figure (b). Answer the following questions. a. In one sentence, state the objective of experiment referring to Fig a. b. What can you conclude from bottom panel of figure a where ErbB2 antibody is being used? c. In one sentence, state the objective of experiment referring to Fig b. d. What can you conclude based on data of figure b?
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Nucleus and nuclear transport problem set ErbB-2 is a receptor found on the plasma membrane of mammalian cells. Recent studies have shown that this protein can move from the plasma membrane to the nucleus. Because most proteins that shuttle between the cytoplasm and the nucleus are soluble and not integral membrane proteins, the mechanism by which ErbB2 undergoes this transport is of particular interest. In the first experiment, RNA interference (siRNA) is used to reduce levels of importin ̢1. Cells are either not subjected to siRNA (-), or have their expression of importin ̢1 reduced (imp), or are subjected to a nonsense siRNA (NS). The presence of ErbB-2 in both cytoplasmic and nuclear fractions is then analyzed by immunoblotting using antibodies against each of these proteins. The data are shown on the immunoblot below in Figure (a). In the second experiment, lysates of cells expressing either wild-type (WT) ErbB-2 or mutant ErbB-2 lacking its nuclear localization signal (ΔNLS) are immunoprecipitated with antibody to ErbB-2 or a control antibody (mIgG). The immunoprecipitates are then examined by immunoblotting with antibodies to importin ̢1 or to ErbB-2 as shown in Figure (b). Answer the following questions; 1a) What is the objective of experiment referring to Fig a? b) What can you conclude from top panel of figure a where importin antibody is being used? c) What can you conclude from bottom panel of figure a where ErbB2 antibody is being used? d) What is the objective of experiment referring to Fig b? e) What can you conclude from figure b?
Sri K.
Modified Figure 2A In the adapted version of Figure 2A above, human cell lines were infected with either wild-type KSHV, or other mutant forms of KSHV (KSHV-K1₁ጃጢጢ, KSHVΔK1, KSHV-K1ጱጱጵ, KSHV-K1₅ₓₑₒₑ) as indicated in the figure. Three Western blots are shown from these cells. • Dox indicates the presence of doxycycline, a drug that initiates viral production in these cells • pAkt (S473) is the activated human Akt signaling protein, which is phosphorylated at serine 473 when activated • vIL-6 is an acronym for viral interleukin-6, a protein encoded by KSHV • Tubulin is a protein is that thought to be expressed at constant levels, under all conditions. 9. (2 points) How many primary antibodies were used to develop this adapted figure, and what are they specific for? 10. (2 points) Given this Western blot, which versions of KSHV can activate human Akt upon KSHV infection? 11. (2 points) What is the specific purpose for detecting tubulin, and why do the investigators wish to detect it? Explain in three sentences at most. 12. (2 points) What is the purpose of the sample that contains no doxycycline? Why did the investigators include this lane? Explain in three sentences at most.
Analyze the Data 7-3: The Ins and Outs of Cell Membranes Two membrane preparations have been derived from the plasma membrane of erythrocytes and are present in a buffer solution: (1) right-side-out membrane vesicles, in which the exoplasmic leaflet of the vesicle faces the buffer (equivalent in orientation to that of the intact plasma membrane); and (2) inside-out membrane vesicles in which the exoplasmic leaflet faces the interior of the vesicles. Right-side-out vesicles (ROVs) Inside-out vesicles (IOVs) a. Annexin V is a protein that binds to phosphatidylserine, one of the phospholipids present in cell membranes. Fluorescently labeled annexin V is mixed with each vesicle preparation. The vesicles are washed to remove unbound annexin V and then examined by fluorescence microscopy. Presence of annexin V is detected by green fluorescence, as shown above. What can be deduced from these data? b. A monoclonal antibody has been prepared against band 3, an integral membrane protein of the erythrocyte plasma membrane. The antibody recognizes an epitope on the carboxy terminus of band 3. Incubation of the two membrane vesicle preparations with this antibody following by staining with a fluorescently labeled secondary antibody generates data similar to that shown in the micrographs above. What can you deduce about the structure of band 3 from these data? c. The two vesicle fractions are either untreated (-) or are incubated with the protease trypsin (+). The membrane vesicles are then washed, the membranes solubilized and the proteins denatured and analyzed by SDS gel electrophoresis and Western blotting using polyclonal antibodies against glycophorin A, an erythrocyte plasma membrane protein. The following data are obtained: ROV IOV ROV IOV Trypsin: - - + + What can be deduced from these data about the structure of glycophorin A?
Dominador T.
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