star-igg1  (Altor BioScience)

Journal: EMBO Molecular Medicine

Article Title: The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

doi: 10.15252/emmm.202113952

Figure Lengend Snippet: A Representative confocal microscopy of 10‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). See also Fig . B Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). C, D Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w 1118 wild‐type ( Droj2 +/+ ) and knockdown ( Droj2 +/− ) 10‐day‐old male flies. Dot plots show all data points along with the mean (bar) ± SD n = 12. * P < 0.05. Unpaired, two‐tailed t ‐test. See also Fig . E, F qPCR analysis of Droj2‐mRNA levels of 3‐ to 6‐day‐old female (E) and male (F) flies expressing human Abeta42 (UAS‐A42) of w 1118 wild‐type flies ( Droj2 +/+ ) normalized to corresponding isogenic w 1118 wild‐type flies without Abeta42 expression ( Droj2 +/+ ctrl). Reference gene is Rpl32 . Dot plots show all data points along with the mean (line) ± SD n = 3. ** P < 0.01. One sample t ‐test against 1. G Immunoprecipitation (IP: 6E10) of synthetic Abeta42 added to the fly head extract of w 1118 wild‐type ( Droj2 +/+ ) flies (Input). Abeta‐specific antibody 6E10 was used for Abeta42‐ and DnaJA1‐specific antibody for Droj2 immunoblot (WB) detection. Showing input, supernatant, supernatant after washing step (WS), and eluate. See also Fig . Source data are available online for this figure.

Article Snippet: These primary antibodies were detected with the following fluorophore‐conjugated secondary antibodies for gSTED microscopy: STAR RED FluoTag‐X2 sdAb anti‐Mouse IgG1 (Clone 10A4, Nanotag, N2002‐AbRED‐S, 1:500), Alexa Fluor 594 Goat anti‐Mouse IgG2b (Invitrogen, A‐21145, 1:100), or Alexa Fluor 594 Goat anti‐Mouse IgG2a (Invitrogen, A‐21135, 1:100).

Techniques: Confocal Microscopy, Staining, Knockdown, Expressing, Two Tailed Test, Immunoprecipitation, Western Blot

Journal: EMBO Molecular Medicine

Article Title: The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

doi: 10.15252/emmm.202113952

Figure Lengend Snippet: A, B Survival of female (A) and male (B) w 1118 wild‐type flies ( Droj2 +/+ ) and Droj2 knockdown flies ( Droj2 +/− ) with expression of human Abeta42 (UAS‐A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl 2 . Survival was determined at indicated time points. n = 6 with 100–120 flies per experiment. The indicated P ‐value refers to the interaction (int.) term of a Cox proportional hazards model comparing Abeta42 toxicity (UAS‐A42 versus ctrl) and Droj2 expression ( Droj2 +/+ versus Droj2 +/− ) as main factors. The following pairwise comparisons of the indicated groups survival were done by log rank test (**** P < 0.0001; ns, P > 0.05). See also Appendix Fig –K. C Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) both expressing human Abeta42 (UAS‐A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± SD n = 6. * P < 0.05. Unpaired, two‐tailed t ‐test. See also Fig . D Representative gSTED deconvolved images of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A‐specific antibody (ATP5A, green) of Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). E, F Solidity (E) and circularity (F) normalized to corresponding wild‐type Droj2 +/+ control (without Abeta42 expression, dashed line) of ATP5A‐stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8 to 10 brains of w 1118 wild‐type ( Droj2 +/+ ) and knockdown ( Droj2 +/− ) 15‐day‐old male flies expressing human Abeta42 (UAS‐A42). Dot plots show all data points along with the mean (bar) ± SD n = 8–10. * P < 0.05. Unpaired, two‐tailed t ‐test. See also Fig .

Article Snippet: These primary antibodies were detected with the following fluorophore‐conjugated secondary antibodies for gSTED microscopy: STAR RED FluoTag‐X2 sdAb anti‐Mouse IgG1 (Clone 10A4, Nanotag, N2002‐AbRED‐S, 1:500), Alexa Fluor 594 Goat anti‐Mouse IgG2b (Invitrogen, A‐21145, 1:100), or Alexa Fluor 594 Goat anti‐Mouse IgG2a (Invitrogen, A‐21135, 1:100).

Techniques: Knockdown, Expressing, Control, Two Tailed Test, Marker, Staining

Journal: EMBO Molecular Medicine

Article Title: The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

doi: 10.15252/emmm.202113952

Figure Lengend Snippet: A Pearson’s correlation coefficient and Manders’ co‐localization Coefficients of endoplasmatic reticulum protein KDEL and Abeta analyzed on confocal images of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 and KDEL‐specific antibody in Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) with expression of human Abeta42 (UAS‐A42). Dot plots show all data points along with the mean (bar) ± SD n = 10. Unpaired, two‐tailed t ‐test. B Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and endoplasmic reticulum protein KDEL‐specific antibody (KDEL, green) of Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). C Representative confocal and gSTED deconvolved (decon) microscopy of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A‐specific antibody (ATP5A, green) of w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). D–H Counts of mitochondria (D), mitochondria average size (E), mitochondria coverage area (F), Feret diameter (G), and min Feret diameter (H) of ATP5A‐stained mitochondria from fly brain gSTED deconvolved images representatively shown in Fig from 9 to 10 brains of w 1118 wild‐type ( Droj2 +/+ ) and knockdown ( Droj2 +/− ) male flies expressing human Abeta42 (UAS‐A42). Dot plots show all data points along with the mean (bar) ± SD n = 9–10. Unpaired, two‐tailed t ‐test or Mann–Whitney test.

Article Snippet: These primary antibodies were detected with the following fluorophore‐conjugated secondary antibodies for gSTED microscopy: STAR RED FluoTag‐X2 sdAb anti‐Mouse IgG1 (Clone 10A4, Nanotag, N2002‐AbRED‐S, 1:500), Alexa Fluor 594 Goat anti‐Mouse IgG2b (Invitrogen, A‐21145, 1:100), or Alexa Fluor 594 Goat anti‐Mouse IgG2a (Invitrogen, A‐21135, 1:100).

Techniques: Knockdown, Expressing, Two Tailed Test, Microscopy, Marker, Staining, MANN-WHITNEY