Journal: Cell reports

Article Title: The Kinase Activity of Drosophila BubR1 Is Required for Insulin Signaling-Dependent Stem Cell Maintenance.

doi: 10.1016/j.celrep.2020.107794

Figure Lengend Snippet: Figure 1. Flies with Point Mutations That Disrupt BubR1 Functions (A) Schematic of Drosophila BubR1 and the key residues that are mutated in this study. (B) qPCR analysis of BubR1 mRNA abundance in different mutant flies. The two kinase-dead mutants show slight changes in mRNA levels in opposite directions when compared with wild type (WT) (t test, p < 0.05 and p < 0.01, respectively). Error bars represent SEMs; n = 3. (C) Western blot analysis of BubR1 protein levels in WT and mutant flies. (D) Chromosome spreads of third-instar neuro- blasts from WT and BubR1 mutants. (E) While WT and the two kinase-dead mutants have normal chromosome numbers, the BubR1AAN mutant shows elevated chromosomal instability (chi-square test, p < 0.05). Bar: 2 mm. (F) Colchicine injection scheme. (G) Real-time records of histone (RFP-H2AvD) show that colchicine injection leads to prolonged mitotic arrest in WT and BubR1 kinase-dead (BubR1K1204A

Article Snippet: The antibodies used in this studywere as follow: rabbit anti-AKT and anti-pAKT antibodies (CST #4691T and #4060T, 1:1000), rabbit anti-BubR1 antibody (a gift from Claudio Sunkel, 1:2000), mouse anti-tubulin (CST, 1:2000), mouse anti-b-actin (Servicebio, 1:1000), rabbit anti-HA (CST, 1:2000), and mouse anti-Flag (Sigma, 1:2000). e3 Cell Reports 31, 107794, June 23, 2020

Techniques: Mutagenesis, Western Blot, Injection

Journal: Cell reports

Article Title: The Kinase Activity of Drosophila BubR1 Is Required for Insulin Signaling-Dependent Stem Cell Maintenance.

doi: 10.1016/j.celrep.2020.107794

Figure Lengend Snippet: Figure 3. Compromised Insulin Signaling in Flies Lacking BubR1 Kinase Activity (A) BubR1K1204A and BubR1D1326A mutations extend lifespan (log rank test, WT versus BubR1K1204A p = 0.0003, WT versus BubR1D1326A p = 0.0007, WT versus BubR1AAN p = 0.2206). (B) Schematic diagram of the insulin signaling pathway. (C) The PI3K reporter t-GPH (green) shows reduced membrane localization in BubR1K1204A and BubR1D1326A intestines. Bar: 10 mm. (D) Western blot analyses of pAKT and AKT on 12- to 14-day-old WT and mutants flies. (E) Quantifica- tion of the western blot results demonstrates reduced pAKT:AKT ratios in BubR1K1204A and BubR1D1326A flies compared to WT (t test, p < 0.01). Error bars represent SEMs; n = 3. (F) qPCR results showing the expression of Thor, a target gene of dFoxO, in the ovaries of WT and BubR1 mutants. Error bars represent SEMs; n = 3. (G) Staining of InR in the salivary gland. While clear membrane distribution of InR (green) can be observed in WT, this membrane signal of InR is greatly diminished in BubR1 mutants. E-cadherin (purple) is used to visualize the membranes. Bar: 10 mm. See also Figure S3.

Article Snippet: The antibodies used in this studywere as follow: rabbit anti-AKT and anti-pAKT antibodies (CST #4691T and #4060T, 1:1000), rabbit anti-BubR1 antibody (a gift from Claudio Sunkel, 1:2000), mouse anti-tubulin (CST, 1:2000), mouse anti-b-actin (Servicebio, 1:1000), rabbit anti-HA (CST, 1:2000), and mouse anti-Flag (Sigma, 1:2000). e3 Cell Reports 31, 107794, June 23, 2020

Techniques: Activity Assay, Membrane, Western Blot, Expressing, Staining