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. 2021 Apr 28;220(7):e202009179. doi: 10.1083/jcb.202009179

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© 2021 Venkatesan et al.

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Figure S1. — Mutant CRT is multimeric via disulfide bonds in MPN patient platelets. (A) SDS-PAGE (8% gels) under reducing conditions and immunoblots of platelet lysates from healthy donors and MPN patient platelets were probed with the indicated antibodies. The same lysates were loaded onto two gels and probed with anti-CRT(C_mut) (top panel) or anti-CRT(N) (middle panel) antibodies. The latter was reprobed with anti-vinculin (lower panel) antibody. Anti-CRT(C_mut) does not detect WT CRT in healthy control (HC) lysates. Lysate 4995 is a CRT_Ins5 mutant, which based on size (Fig. 2 A), should migrate more slowly than CRT_Del52 (all other samples in the blot). (B) Lysates from MPN patients or healthy donor platelets were separated by reducing or nonreducing SDS-PAGE (4–20% gradient gels), and the same lysates were loaded onto two gels probed with anti-CRT(C_mut) (top panel) or anti-CRT(N) (middle panel) antibodies. The former was reprobed with anti-GAPDH antibody. Monomer and multimer CRT bands are indicated. Bands indicated as dimers or multimers are overrepresented under nonreducing conditions in the CRT mutant platelet lysates, whereas monomer bands are depleted under the same conditions for 8251-2 and 2648-3, which appear to have a high mutational load. The lower expression of mutant CRT in 2028-1 patient lysate precluded its detection under nonreducing conditions at greater than background levels. Anti-CRT(C_mut) does not detect WT CRT in HC lysates; hence, no bands are visualized in lanes labeled HC in immunoblots with anti-CRT(C_mut). Nonspecific bands that are detected in the reducing blots are marked as such. (C and D) Native immunoblots (8% gels) of platelet lysates (25 µg) from MPN patient platelets or same-day healthy donor platelets or RBC lysates were probed with anti-CRT(C_mut) (left panels) and anti-CRT(N) (right panels) antibodies. Oligomeric forms of mutant CRT are readily detectable at the top of the gel, particularly with samples such as 2648-2, which showed high expression of mutant CRT. Boxes indicate hemoglobin (Hb) contamination of platelets, verified using RBC lysates. CRT HC indicates HC samples; CRT mutant patient samples are indicated as Del52 or Ins5; JAK2 indicates JAK2 mutant patient samples; and RBC indicates RBC lysates. MW, molecular weight.