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KNOLLE as part of SNARE complex (E>>G:SNAP33/ big3 , estradiol-induced, +EST) either trapped at TGN (+ brefeldin A, <t>BFA)</t> or trafficked to the cell division plane (-BFA) was tested for ubiquitination. Lines for comparison: Con, non-transgenic control; G:KN, GFP-tagged KNOLLE.Protein extracts from seedlings treated with wortmannin (Wm) and NEM (+Wm/NEM) or untreated (-Wm/NEM) were immunoprecipitated with the GFP trap (IP(α-GFP)) and the enriched fractions probed with anti-GFP, anti-ubiquitin and anti-KNOLLE antibodies; bottom panel, prolonged exposure. Expected sizes: GFP:KNOLLE, 65 kDa; (UBQ)n-GFP:KNOLLE, 80-120 kDa (UBQ moiety 15-60 kDa); endogenous KNOLLE, 33 kDa; (UBQ)n-KNOLLE, 50-100 kDa. Brackets, ubiquitinated bands of GFP:KNOLLE; wide braces, range of (UBQ)n of KNOLLE between 50 and 100 kDa; narrow braces, the highest (UBQ)n band of KNOLLE at approximately 100 kDa. Asterisk, GFP:KNOLLE; double asterisks, GFP:SNAP33; hashtags, cross-reacting bands of anti-KNOLLE antiserum; numbers on the left, protein sizes in kDa.
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KNOLLE as part of SNARE complex (E>>G:SNAP33/ big3 , estradiol-induced, +EST) either trapped at TGN (+ brefeldin A, <t>BFA)</t> or trafficked to the cell division plane (-BFA) was tested for ubiquitination. Lines for comparison: Con, non-transgenic control; G:KN, GFP-tagged KNOLLE.Protein extracts from seedlings treated with wortmannin (Wm) and NEM (+Wm/NEM) or untreated (-Wm/NEM) were immunoprecipitated with the GFP trap (IP(α-GFP)) and the enriched fractions probed with anti-GFP, anti-ubiquitin and anti-KNOLLE antibodies; bottom panel, prolonged exposure. Expected sizes: GFP:KNOLLE, 65 kDa; (UBQ)n-GFP:KNOLLE, 80-120 kDa (UBQ moiety 15-60 kDa); endogenous KNOLLE, 33 kDa; (UBQ)n-KNOLLE, 50-100 kDa. Brackets, ubiquitinated bands of GFP:KNOLLE; wide braces, range of (UBQ)n of KNOLLE between 50 and 100 kDa; narrow braces, the highest (UBQ)n band of KNOLLE at approximately 100 kDa. Asterisk, GFP:KNOLLE; double asterisks, GFP:SNAP33; hashtags, cross-reacting bands of anti-KNOLLE antiserum; numbers on the left, protein sizes in kDa.
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KNOLLE as part of SNARE complex (E>>G:SNAP33/ big3 , estradiol-induced, +EST) either trapped at TGN (+ brefeldin A, <t>BFA)</t> or trafficked to the cell division plane (-BFA) was tested for ubiquitination. Lines for comparison: Con, non-transgenic control; G:KN, GFP-tagged KNOLLE.Protein extracts from seedlings treated with wortmannin (Wm) and NEM (+Wm/NEM) or untreated (-Wm/NEM) were immunoprecipitated with the GFP trap (IP(α-GFP)) and the enriched fractions probed with anti-GFP, anti-ubiquitin and anti-KNOLLE antibodies; bottom panel, prolonged exposure. Expected sizes: GFP:KNOLLE, 65 kDa; (UBQ)n-GFP:KNOLLE, 80-120 kDa (UBQ moiety 15-60 kDa); endogenous KNOLLE, 33 kDa; (UBQ)n-KNOLLE, 50-100 kDa. Brackets, ubiquitinated bands of GFP:KNOLLE; wide braces, range of (UBQ)n of KNOLLE between 50 and 100 kDa; narrow braces, the highest (UBQ)n band of KNOLLE at approximately 100 kDa. Asterisk, GFP:KNOLLE; double asterisks, GFP:SNAP33; hashtags, cross-reacting bands of anti-KNOLLE antiserum; numbers on the left, protein sizes in kDa.
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KNOLLE as part of SNARE complex (E>>G:SNAP33/ big3 , estradiol-induced, +EST) either trapped at TGN (+ brefeldin A, BFA) or trafficked to the cell division plane (-BFA) was tested for ubiquitination. Lines for comparison: Con, non-transgenic control; G:KN, GFP-tagged KNOLLE.Protein extracts from seedlings treated with wortmannin (Wm) and NEM (+Wm/NEM) or untreated (-Wm/NEM) were immunoprecipitated with the GFP trap (IP(α-GFP)) and the enriched fractions probed with anti-GFP, anti-ubiquitin and anti-KNOLLE antibodies; bottom panel, prolonged exposure. Expected sizes: GFP:KNOLLE, 65 kDa; (UBQ)n-GFP:KNOLLE, 80-120 kDa (UBQ moiety 15-60 kDa); endogenous KNOLLE, 33 kDa; (UBQ)n-KNOLLE, 50-100 kDa. Brackets, ubiquitinated bands of GFP:KNOLLE; wide braces, range of (UBQ)n of KNOLLE between 50 and 100 kDa; narrow braces, the highest (UBQ)n band of KNOLLE at approximately 100 kDa. Asterisk, GFP:KNOLLE; double asterisks, GFP:SNAP33; hashtags, cross-reacting bands of anti-KNOLLE antiserum; numbers on the left, protein sizes in kDa.

Journal: bioRxiv

Article Title: Degradation of cytokinesis-specific Qa-SNARE KNOLLE is regulated by context-dependent ubiquitination

doi: 10.64898/2026.05.13.724867

Figure Lengend Snippet: KNOLLE as part of SNARE complex (E>>G:SNAP33/ big3 , estradiol-induced, +EST) either trapped at TGN (+ brefeldin A, BFA) or trafficked to the cell division plane (-BFA) was tested for ubiquitination. Lines for comparison: Con, non-transgenic control; G:KN, GFP-tagged KNOLLE.Protein extracts from seedlings treated with wortmannin (Wm) and NEM (+Wm/NEM) or untreated (-Wm/NEM) were immunoprecipitated with the GFP trap (IP(α-GFP)) and the enriched fractions probed with anti-GFP, anti-ubiquitin and anti-KNOLLE antibodies; bottom panel, prolonged exposure. Expected sizes: GFP:KNOLLE, 65 kDa; (UBQ)n-GFP:KNOLLE, 80-120 kDa (UBQ moiety 15-60 kDa); endogenous KNOLLE, 33 kDa; (UBQ)n-KNOLLE, 50-100 kDa. Brackets, ubiquitinated bands of GFP:KNOLLE; wide braces, range of (UBQ)n of KNOLLE between 50 and 100 kDa; narrow braces, the highest (UBQ)n band of KNOLLE at approximately 100 kDa. Asterisk, GFP:KNOLLE; double asterisks, GFP:SNAP33; hashtags, cross-reacting bands of anti-KNOLLE antiserum; numbers on the left, protein sizes in kDa.

Article Snippet: Estradiol induction and brefeldin A (BFA) treatment: 50 μM BFA (50 mM stock in DMSO/Ethanol, Invitrogen) was applied for 30 minutes, followed by 20 μM estradiol (20 mM stock in DMSO, Sigma-aldrich) for 3 hours and 30 minutes in the presence of BFA and subsequently wortmannin and NEM were added as described above.

Techniques: Ubiquitin Proteomics, Comparison, Transgenic Assay, Control, Immunoprecipitation