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. 2003 Aug;23(16):5581–5593. doi: 10.1128/MCB.23.16.5581-5593.2003

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FIG. 8. — NΔCterm promotes accumulation of ubiquitinated N and SuH fragments. (a) Increased expression of N^1791-2155 in clone 8 cells results in a stronger loss of NFull and SuH than in S2-NFull cells. Compare lanes 3 and 4 with lanes 1 and 2. Equal amounts of total proteins were loaded in lanes 1 and 2, and in lanes 3 and 4. The band marked with an asterisk serves as an indicator of relative loading. Proteins were electrophoresed in SDS-8% PAGE gels. (b) N^1791-2155 promotes accumulation of ubiquitinated N (Ubi-N*) and a smaller SuH fragment (SuH*) that is ubiquitinated (Ubi-SuH*; see c below). Compare lanes 2 and 4 with lanes 1 and 3 for N; lanes 6 and 8 with lanes 5 and 7 for SuH. Panels 5*, 6*, 7*, and 8* are magnified (×2) images of relevant portions of panels 5 to 8. The blot in lanes 1 and 2 was reprobed for lanes 3 and 4, the blot in lanes 5 and 6 for lanes 7 and 8. Lanes 9 and 10 is the blot of supernatants of SuH immunoprecipitates indicating the levels of total proteins in extracts. The blot of supernatants of N immunoprecipitates was almost identical (not shown). Proteins were electrophoresed in SDS-4% PAGE gels. (c) N^1-2155 also promotes accumulation of Ubi-SuH* (lanes 3 and 4). Only a fraction of SuH* molecules appear to be ubiquitinated as the amount of SuH* was similar in S2 and S2-N^1-2155 cells (lanes 5 and 6). Lanes 1 and 2 indicate the total amount of proteins in the extracts used for lanes 3 and 4. Lanes 5 and 6 show the blot from lanes 3 and 4 reprobed with an SuH antibody. Proteins were electrophoresed in SDS-8% PAGE gels. (d) Treatment of S2-N^1-2155 cells with 26 S proteasome inhibitors (M115 + lactacystin) promotes accumulation of ubiquitinated proteins as expected (lanes 1 and 2) but loss of SuH proteins (lanes 3 and 4). SuH is recognized by polyclonal rat αSuH (r) from F. Schweisguth, by polyclonal mouse αSuH (m) from S. Kidd and T. Lieber, by polyclonal rat αSuH (r1) generated by us (three different animals), and by the monoclonal rat αSuH antibody from S. Artavanis-Tsakonas. SuH* is recognized weakly by αSuH (r) and αSuH (m); the latter more so than the former. The band marked with an asterisk is also recognized by αSuH (m) (strongly) and αSuH (r) (weakly). The band marked with # is also recognized weakly by αSuH (r). The remaining bands shown in lane 4 are associated with heat shock induced SuH. Whether or not they are SuH fragments or related proteins is unknown. PIs, protease inhibitors.

FIG. 8. — NΔCterm promotes accumulation of ubiquitinated N and SuH fragments. (a) Increased expression of N^1791-2155 in clone 8 cells results in a stronger loss of NFull and SuH than in S2-NFull cells. Compare lanes 3 and 4 with lanes 1 and 2. Equal amounts of total proteins were loaded in lanes 1 and 2, and in lanes 3 and 4. The band marked with an asterisk serves as an indicator of relative loading. Proteins were electrophoresed in SDS-8% PAGE gels. (b) N^1791-2155 promotes accumulation of ubiquitinated N (Ubi-N*) and a smaller SuH fragment (SuH*) that is ubiquitinated (Ubi-SuH*; see c below). Compare lanes 2 and 4 with lanes 1 and 3 for N; lanes 6 and 8 with lanes 5 and 7 for SuH. Panels 5*, 6*, 7*, and 8* are magnified (×2) images of relevant portions of panels 5 to 8. The blot in lanes 1 and 2 was reprobed for lanes 3 and 4, the blot in lanes 5 and 6 for lanes 7 and 8. Lanes 9 and 10 is the blot of supernatants of SuH immunoprecipitates indicating the levels of total proteins in extracts. The blot of supernatants of N immunoprecipitates was almost identical (not shown). Proteins were electrophoresed in SDS-4% PAGE gels. (c) N^1-2155 also promotes accumulation of Ubi-SuH* (lanes 3 and 4). Only a fraction of SuH* molecules appear to be ubiquitinated as the amount of SuH* was similar in S2 and S2-N^1-2155 cells (lanes 5 and 6). Lanes 1 and 2 indicate the total amount of proteins in the extracts used for lanes 3 and 4. Lanes 5 and 6 show the blot from lanes 3 and 4 reprobed with an SuH antibody. Proteins were electrophoresed in SDS-8% PAGE gels. (d) Treatment of S2-N^1-2155 cells with 26 S proteasome inhibitors (M115 + lactacystin) promotes accumulation of ubiquitinated proteins as expected (lanes 1 and 2) but loss of SuH proteins (lanes 3 and 4). SuH is recognized by polyclonal rat αSuH (r) from F. Schweisguth, by polyclonal mouse αSuH (m) from S. Kidd and T. Lieber, by polyclonal rat αSuH (r1) generated by us (three different animals), and by the monoclonal rat αSuH antibody from S. Artavanis-Tsakonas. SuH* is recognized weakly by αSuH (r) and αSuH (m); the latter more so than the former. The band marked with an asterisk is also recognized by αSuH (m) (strongly) and αSuH (r) (weakly). The band marked with # is also recognized weakly by αSuH (r). The remaining bands shown in lane 4 are associated with heat shock induced SuH. Whether or not they are SuH fragments or related proteins is unknown. PIs, protease inhibitors.