htt antibodies 2b7  (Bio-Rad)

Journal: Nature Communications

Article Title: An alternative splicing modulator decreases mutant HTT and improves the molecular fingerprint in Huntington’s disease patient neurons

doi: 10.1038/s41467-022-34419-x

Figure Lengend Snippet: a Paradigm illustrating the HD patient-based disease model (fibroblasts, iPSC, cortical progenitors (25d old), and cortical neurons (35d old)) and readouts. Created with BioRender.com. b Bar plot depicting FACS quantification of NESTIN/PAX6 double-positive cells. Statistics: Welch’s test. Bars: median ± IQR. c Bar plot illustrating FACS quantification of bIII-Tubulin/CTIP2 double-positive cells. Statistics: Welch’s test. Bars: median ± IQR. d Representative pictures of cortical neurons. Scale bar 50 µm. e Illustration depicting the MSD HTT quantification assay, where the added protein samples bind to 2B7 antibody, used for coating the plates. The SULFO-TAG coupled antibodies D7F7 and MW1 are added for quantification of total HTT and mutant HTT, respectively. Note: numeric values from 2B7/D7F7 assay (total HTT) cannot be directly set in relation to numeric values from 2B7/MW1 assay (mutant HTT). Created with BioRender.com. f Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in fibroblasts (4 Ctrl lines, 4 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test ( P value = 0.9717); mHTT Welch’s test ( P value = 0.0737). Bars: median ± IQR. g Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in iPSC (8 Ctrl lines, 9 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test ( P value = 0.2080); mHTT Mann–Whitney test ( P value < 0.0001). Bars: median ± IQR. h Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in cortical progenitors (7 Ctrl lines, 9 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test ( P value = 0.2149); mHTT Welch’s test ( P value = 0.0061). Bars: median ± IQR. i Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in cortical neurons (7 Ctrl lines, 8 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test ( P value = 0.2781); mHTT Welch’s test ( P value = 0.0239). Bars: median ± IQR. Source data are provided as a Source Data file.

Article Snippet: The MSD assay plate was coated with 5ug/ml of the N-terminally binding HTT antibody 2B7 (#CH03023, Coriell) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3) overnight.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: eLife

Article Title: A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

doi: 10.7554/elife.05449

Figure Lengend Snippet: Figure 1. Gpr52 modulates Htt levels. All data plots: average and S.E.M.; ‘*’: p < 0.05, ‘**’: p < 0.01, ‘***’: p < 0.001 by the two-tailed Mann–Whitney U test. The number on top of each bar indicates the biological replicate number. (A) Transfection of Gpr52 siRNAs (Gpr52_si1∼3) in the mouse striatal cells (STHdhQ7/Q111) lowers Htt levels, as detected by Htt antibodies MW1, 2166, ab1 and 2B7. MW1 is the polyQ antibody that detects only the mHtt protein, whereas 2166, 2B7 and ab1 detects both mHtt and wtHtt. Left panels: representative western-blots; Hdh5 is the Htt siRNA used as the positive control for Htt knock-down. Neg is the non-targeting siRNA used as the negative control. Right panel: western-blot quantification from multiple replicates. (B) Infection of lentiviruses expressing Gpr52 shRNAs (Gpr52_sh1∼2) lowers Htt in primary striatal but not cortical neurons cultured from HdhQ140/Q140 knock-in mice. Left panels: representative western-blots. Right panel: western-blot quantification for the normalized 3B5H10 signals from multiple replicates. (C) Heterozygous knockout of Gpr52 lowers Htt in vivo in the striata but not cortices of HdhQ140/Q7 knock-in mice in vivo. The mice were obtained by crossing the heterozygous Gpr52 knockout mice with the HdhQ140/Q140 knock-in mice. Littermates between 40 to 69 days of age were analyzed. Left panels: representative western-blots. Right panel: western-blot quantification of the normalized MW1 signals from multiple mouse samples. Each dot represents the signal from a single mouse. (D) Left panels: Immunostaining of HD patient iPS- derived striatal-like neurons. Differentiated neurons from HD patient’s iPS cells express molecular markers for striatal medium spiny neurons: Tuj1, GABA and DARPP32. Scale bar: 50 μM. Right panels: Transfection of human Gpr52 siRNAs (hGpr52_si1∼2) in the HD patient iPS-derived neurons lowers Htt levels detected by both western-blots and Figure 1. continued on next page

Article Snippet: Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 (Weiss et al., 2009) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4 ̊C.

Techniques: Two Tailed Test, MANN-WHITNEY, Transfection, Western Blot, Positive Control, Knockdown, Negative Control, Infection, Expressing, Cell Culture, Knock-In, Knock-Out, In Vivo, Immunostaining, Derivative Assay

Journal: eLife

Article Title: A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

doi: 10.7554/elife.05449

Figure Lengend Snippet: Figure 3. Gpr52 modulates Htt levels via cAMP dependent but PKA independent pathways. All experiments are performed in the mouse striatal cell line STHdhQ7/Q111, and all data are plotted as average and S.E.M. ‘*’: p < 0.05, ‘**’: p < 0.01, ‘***’: p < 0.001. The number on top of each bar indicates the biological replicate number. (A) Changes of cAMP levels measured by the cAMP-Glo assay (Promega). Gpr52 siRNAs were tranfected for 3 days, whereas the compound treatment (forskolin: 1 μM; reserpine: 10 μM) lasts for 24 hr; statistical analyses performed by the two- tailed Mann–Whitney U test. (B) Htt level measured by the 2B7/2166 HTRF (Liang et al., 2014) upon treatment of Figure 3. continued on next page

Article Snippet: Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 (Weiss et al., 2009) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4 ̊C.

Techniques: Glo Assay, Two Tailed Test, MANN-WHITNEY