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. Author manuscript; available in PMC: 2014 Jul 3.
Published in final edited form as: Cell. 2013 Jul 3;154(1):118–133. doi: 10.1016/j.cell.2013.05.059

Figure 1. The C terminal fragment of α1A subunit initiates at MIMEY (amino acids 1960-1964, nucleotide 6114-6128).

Figure 1

(A) Western blot analysis of fractions collected from HiTrap™ DEAE FF anion exchange chromatography. 3xFLAG-tagged α1A subunit is used as positive control. (B) Coomassie blue staining of the peak α1ACT-containing fraction after two-step purification. The arrowhead indicates the 75 kD band identified by mass spectrometry as α1ACT. (C) Western blot analysis of lysate from α1A overexpressing cells with anti-FLAG antibody confirms the identity of α1ACT. (D) LC-MS/MS analysis of in-gel digest of proprionylated protein reveals that the starting amino acid sequence of N terminus of the α1ACT fragment is Met Ile Met Glu Tyr. (E) Schematic representation of the constructs with a series of mutations or deletions. (F) In-frame deletion of the start site of α1ACT does not abolish the expression of the 75kD C terminal portion of the FLAG-tagged α1A protein bearing either normal range (Q11) or pathological range (Q33) of polyQ. (G) Expression of the 75kD α1ACT C terminal fragment persists after insertion of termination codons at T1937 or P1847 in the FLAG-tagged α1A subunit, upstream of the start site. (H) Deletion of a 534bp fragment (α1Adel534Q11), but not deletion of a 185bp fragment (α1Adel185Q11) from the α1A coding region upstream of the α1ACT eliminates α1ACT expression, while maintaining expression of full-length α1A-FLAG. Deletions using encoded Q33 repeat expansions constructs (α1Adel185Q33 and α1Adel534Q33) behave similar to the Q11 constructs (see also Figure S1).