Fig 2: PLTFBH selectively decreases protein levels of certain members of HSP40/JDPs. (A) Western blotting for several members of HSP40/JDPs and GAPDH using HN31 cells treated with DMSO (D) or PLFBH (TF) at ~1/2 IC50 for 24 h. (B) Three groups (good, moderate, little or no) of HSP40/JDPs based on their response to PLTFBH. Note that some blots were from the same membrane (DNAJB1 and DNAJB2; DNAJB6 and DNAJC6; DNAJC2 and DNAJC3), hence using the same GAPDH control. (C) Amino acid sequence alignment of the J domain of multiple HSP40/JDPs by centering the HPD sequence. (D) CETSA showing intracellular binding of PLTFBH with DNAJA4, but not DNAJC6, in CAL33 cells treated with 80 µM of PLTFBH (TF) for 4 h. Vinculin was used as a loading control. Summarized graphs showing normalized DNAJA4 and DNAJC6 band densities at different temperatures (right). Mean ± SEM from three independent experiments (n = 3). * p < 0.05 for two-way ANOVA.

Fig 3: Pedigree, DNA sequencing and schematic representation of DNAJB2 protein domain structure. (a) Family pedigree. (b) Sanger chromatograms of patient II.4 with the homozygous (HOM) c.145delG variant in the DNAJB2 gene, patient II.3 (heterozygous [HET]) and II.5 (wild-type [WT]). (c) DNAJB2 protein domains, the alternatively spliced C-terminal parts of the two isoforms and the residue harboring the disease mutation (p. Val49TrpfsTer25) are indicated. The J, G/F common region, and C-terminal domains (CTD), the Ser/Thr-rich region, two ubiquitin interacting motifs (UIM) and the C-terminal geranylgeranyl (GG) anchor of DNAJB2b are shown. Partly modified from Sarparanta et al., Int J Mol Sci. 2020; 21: 1409