Fig 1: HTT is a part of the TCR complex.(A) Nuclear extract (NE), and cytosolic extract (CE) were purified from human neuroblastoma SH-SY5Y cells and the protein fractions were analyzed by western blots (WBs) to detect HTT, ATXN3, PNKP, and HAP1 levels in these sub-cellular fractions. GAPDH and hnRNPC1/C2 were used as cytosolic and nuclear markers respectively. APE1 was used as a negative control in panels A to D. (B) Endogenous HTT was immunoprecipitated (IP’d) from NEs of SH-SY5Y cells and immunocomplex (IC) were analyzed by western blot (WB) to examine the TCR proteins (HAP-1, ATXN3, PNKP, POLR2A, LIG 3, CBP, and TAFII 130 (TAF4). (C) Endogenous ATXN3 was IP’d from NEs of SH-SY5Y cells and IC was subjected to WB to detect associated TCR complex components with respective antibodies. (D) Endogenous PNKP was IP’d from NEs of SH-SY5Y cells and IC was analyzed by WB to examine associated TCR components. (E) Endogenous POLR2A was IP’d from NEs of SH-SY5Y cells and IC was analyzed by WB to detect associated TCR proteins. (F) NEs was isolated from PC12 cells ectopically expressing a Myc-tagged full-length normal wild type HTT (FL-wtHTT-Q23) for assessing the possible interaction of HTT with POLR2A and associated TCR proteins. Exogenous Myc-wtHTT-Q23 was IP’d with an anti-Myc antibody, and the Myc immunocomplex was subjected to WBs with respective antibodies. APE1 was used as a negative control in panels F and G. (G) NEs was isolated from PC12 cells ectopically expressing a Myc-tagged full-length mutant HTT (FL-mHTT-Q148). Exogenous Myc-wtHTT-Q148 was IP’d with an anti-Myc antibody, and the Myc immunocomplex was subjected to WBs with respective antibodies. Proximity Ligation Assay (PLA) in SH-SY5Y cells to examine the protein-protein interaction using the following antibody pairs. Red fluorescence indicates positive PLA signals for protein-protein interactions. Nuclei were stained with DAPI. (H) ATXN3 (rabbit: Rb) and IgG (mouse: mo) or PNKP (mouse: mo) antibodies. (I) ATXN3 (rabbit: Rb) with IgG (mouse: mo) or HAP-1 (mouse: mo) antibodies. (J) HAP-1 (rabbit: Rb) and IgG (mouse: mo) or HTT (mouse: mo) antibodies. (K) HTT (mouse: mo) with IgG (rabbit: Rb) or ATXN3 (rabbit: Rb) antibodies. (L) HTT (mouse: mo) with IgG (rabbit: Rb) or PNKP (rabbit: Rb) antibodies, and. (M) POLR2A (mouse: mo) with IgG (rabbit: Rb) or HTT (rabbit: Rb) antibodies.