Fig 2: Performance of the standard curve detection by all antibody pairs. The five standard HTT proteins (N573 Q22, N573 Q45, N573 Q72, FL Q17, FL Q46) were serially diluted from 8 pM and assayed via SMC using the antibody pair reported on the title of each chart; the first mentioned antibody was used as capture whereas the second was used as detection. Each experimental point is reported as the average and standard deviations of a triplicate.

Fig 3: Dilution linearity and spike recovery in wild type and BACHD rat brain homogenates. Grey crossed cells represent not tested antibody pairs, black cells are antibody pairs that did not meet the 67% acceptance criteria (see text), green cells show antibody pairs with achieved dilution linearity and/or spike recovery. (a) Two wild type rats(13272 and 13271) and two BACHD rats (13270 and 13266) brain homogenates were diluted starting 1.3 µg/ml of total protein for the 2B7-MW1 assay and from 6.7 µg/ml of total protein for all other antibody pairs for 7 dilution points. Each dilution curve was assayed via SMC using the antibody pair reported on the x-axis. The percentage of the dilution points within ± 20% RE% is reported in each cell of the heat-map. The acceptance threshold is 66.7%; all passed conditions are coloured in green. All data for each matrix and assay is reported in the supplementary figure S-4 along with an example of the dilution linearity calculations. The Dilution linearity of wild type homogenates for the 2B7-MW1, 2B7-HDB4E10, MW1-MAB2166, and MW1-HDB4E10 are not reported as most dilution points were below the LLoQ for these assays. (b) The same four rat brain samples were spiked with a serial dilution of the recombinant FL HTT Q46 and assayed via SMC using the antibody pair reported on the x-axis. The percentage of the recovered spikes, within one matrix, are in each cell of the heat-map. The acceptance threshold is 66.7%; all passed conditions are coloured in green. All data for each matrix and assays are reported in the supplementary figure S-5. The spike recovery test was not run in BACHD rat homogenates with assays including the MW1 antibody as the signal of the endogenous analyte was too high.

Fig 4: Lowering Gpr52 rescues human HD neurons and the in vivo fly HD models.(A) Caspase-glo of STHdhQ7/Q111 (HD) or STHdhQ7/Q7 cells (WT) with indicated transfections (Neg: the non-targeting controls siRNA; Gpr: the Gpr52 siRNA smartpool; Htt: the Htt siRNA Hdh5) and compound treatments; statistical analyses by the two-tailed Mann–Whitney U-test. (B) Caspase 3 activity of patient iPS-derived neurons (Q47) measured by the fluorescent indicator dye before and after BDNF removal (scale bar: 200 µm). Bar plots: quantification of caspase 3 signals corrected by the total cell number (by DAPI) and normalized to the HD controls (second bar). Statistical analyses were performed by the two-tailed Mann–Whitney U-test. (C) Immunostaining of Tuj1 and DAPI showing loss of neurons of HD patient iPS-derived neurons (Q47) cultured under the BDNF-deprived condition with indicated transfections, and the rescue by knocking-down Htt or Gpr52 or Rabgap1l or Rabgap1l + Gpr52 (scale bar: 200 µm). Bar plots: quantification of the area in each field covered by the Tuj1 signal (Tuj1 area) and the nuclei counts. All data normalized to the non-targeting siRNA transfected control samples. Statistical analyses were performed by the two-tailed Mann–Whitney U-test. Sholl plots: the sholl analysis (Sholl, 1953) results plotted for each sample, ‘n’ indicated the number of analyzed neurons. Statistical analysis performed by two-way ANOVA tests. (D) Age-dependent motor performance in normal flies expressing elav-GAL4 driver alone (Negative control, blue dotted lines), HD flies expressing elav-GAL4 driven NT-Htt128Q or FL-Htt200Q alone (black dotted lines), or HD flies crossed to loss of function mutation (LOF) or knock-down (shRNA) lines of Drosophila homologs of Gpr52 or Rabgap1l (red lines). Lowering Gpr52 rescues the motor behavior deficits, whereas lowering Rabgap1l enhances the phenotype. n = 15, statistical analysis performed by one way ANOVA and Dunnett's post-tests.DOI: http://dx.doi.org/10.7554/eLife.05449.014

Fig 5: Screening for modulators of Htt levels in the striatal cells (STHdh).(A) Representative western-blots in the STHdhQ7/Q111 cells of candidate modifiers of mHtt levels. 104 candidate modifiers were selected based on our previous screening results (doi: 10.1038/nn.3367). The genes that had averaged Z score values larger than 1.2 and were not previously identified as validated hits in the patient fibroblasts were selected for testing in STHdh cells by pooled siRNAs (Dharmacon, custom library). The MW1 detected full length mHtt is quantified and normalized to the tubulin signal using the ImageJ software. The siRNAs that change mHtt levels by more than 30% in the same direction in three biological repeats were selected as hits. The number on top of each bar indicates the biological repeat number of siRNA knock-down. (B) The bar plot of mHtt level changes upon knock-down of the identified modifiers. The numbers on top of each bar represent the number of biological replicates tested.DOI: http://dx.doi.org/10.7554/eLife.05449.004