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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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nanopore sequencing  (Plasmidsaurus)
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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transmembrane nanopore  (Novoprotein)
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
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nanopore technology service  (Macrogen)
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Macrogen nanopore technology service
(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by <t>nanopore</t> <t>sequencing</t> and compared across GFP-based expression groups.
Nanopore Technology Service, supplied by Macrogen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by nanopore sequencing and compared across GFP-based expression groups.

Journal: bioRxiv

Article Title: Dissection of Poly(A)-binding protein (PABPC) cellular function using degron-mediated depletion with replacement

doi: 10.64898/2026.06.05.728833

Figure Lengend Snippet: (A) Schematic of the experimental workflow for competitive growth assays, protein-abundance measurements, and tail-length measurements in polyclonal rescue lines. Endogenous PABPC1 and PABPC4 were tagged with mAID for auxin-induced degradation, and rescue variants were expressed from doxycycline-inducible constructs co-expressing GFP. (B) Competitive growth assay tracking GFP fluorescence distributions over time (Day 7, Day 13, Day 21) for (left) WT PABPC1 rescue and (right) PABP1212 rescue. Kernel density plots show shifts in GFP expression, indicating differences in competitive fitness. Median values reported for each density. (C) Immunoblot analysis of WT PABPC1, PABP1212 , and endogenous PABPC-mCherry-mAID levels across GFP expression quartiles, with vinculin as loading control. (D) Poly(A)-tail length distributions for mRNAs of WT PABPC1 rescue and PABP1212 rescue lines, measured in uninduced and sorted GFP-intensity quartiles (bot., midbot, midtop, and top). Tail-length distributions were obtained by nanopore sequencing and compared across GFP-based expression groups.

Article Snippet: Whole plasmid sequencing using nanopore (Plasmidsaurus) confirmed each plasmid was constructed as intended.

Techniques: Quantitative Proteomics, Construct, Expressing, Growth Assay, Fluorescence, Western Blot, Control, Nanopore Sequencing