Journal: The Journal of Biological Chemistry

Article Title: Chronic treatment with the complex I inhibitor MPP + depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)

doi: 10.1074/jbc.RA119.010474

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Densitometry data were normalized to loading control, and the half-lives of PINK1 isoforms were estimated by fitting a one-phase exponential decay curve using Prism 8 software (GraphPad, San Diego, CA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Catalog number Source Dilution PINK1 Rabbit BC100-494 Novus Biological 1:1000 TOM20 Rabbit Sc11415 Santa Cruz 1:10,000 β-ACTIN Mouse A5316 Sigma 1:2000 HSP60 Mouse 611563 BD Transduction 1:1000 BAG6 Mouse Sc365928 Santa Cruz 1:1000 BAG5 Rabbit NB100-56091 Novus Biological 1:1000 BAG2 Mouse Sc101216 Santa Cruz 1:1000 HSP90 Rabbit 4877 Cell Signaling 1:1000 CDC37 Rabbit 3604 Cell Signaling 1:1000 CHIP Rabbit Sc66830 Santa Cruz 1:1000 GAPDH Rabbit Ab37168 Abcam 1:10,000 GFP Rabbit A6455 Invitrogen 1:10,000 V5 Mouse Sc271944 Santa Cruz 1:1000 FLAG Rabbit F7425 Sigma 1:1000 β-Tubulin Rabbit Ab6046 Abcam 1:5000 Open in a separate window List of antibodies used in the study

Techniques: Transduction

Journal: bioRxiv

Article Title: Modulation of neuronal resilience during aging by Hsp70/Hsp90/STI1 chaperone system

doi: 10.1101/258673

Figure Lengend Snippet: A. Representative immunoblots for Heat Shock Factor 1 (HSF1) and Hsp40 in cortical extracts. B - C. Densitometric quantification of HSF1 and Hsp40 protein levels respectively (N = 4/genotype). D. Representative immunoblot for FKBP51 in WT and ΔTPR1 male adult cortical extracts. E. Quantification of FKBP 51 protein levels (N = 5/genotype). F. Representative immunoblots of Hsp90 co-chaperones Aha1, p23, and Cdc37. G-I Densitometric quantification of Aha1, p23 and Cdc37 protein levels respectively (N = 4/genotype). WT (WT/WT), HET (WT/ΔTPR1), ΔTPR1 (ΔTPR1/ΔTPR1). J. Immunoblot of STI1, CHIP and Pin1 in cortical lysates. K. & L. Densitometric quantification of CHIP and Pin1. M. & N. Cyclophilin A (CypA) protein expression in cortical lysates and quantification. (N = 4 for WT and ΔTPR1 and N = 4 for HET for CHIP and N = 8 for all genotypes for Pin1). All data are Mean ± S.E.M. *p < 0.05, **p < 0.01, *** p < 0.0001.

Article Snippet: The primary antibodies used in immunoblotting were: anti-STI1 (1:5000, in-house antibody generated by Bethyl Laboratories Montgomery, USA) , anti-Hsp90 (1:1000, Catalog# 4877, Cell Signalling, Danvers, MA, USA), anti-Hsp70 (1:1000, Catalog# ab2787, Abcam, Cambridge, UK), anti-Hsp40 (1:1000, Catalog# 4868, Cell Signalling, Danvers, MA, USA) anti-CHIP (1:1000, Catalog# 2080, Cell Signalling, Danvers, MA, USA), anti-Glucocorticoid Receptor (1:1000, Catalog# 3660, Cell Signalling, Danvers, MA, USA), anti-HSF1 (1:1000, Catalog# 4356, Cell Signalling, Danvers, MA, USA), anti-Hsp90β (1:1000, Catalog# 5087, Cell Signalling, Danvers, MA, USA), anti-GRK2 (1:1000, Catalog# 3982, Cell Signalling, Danvers, MA, USA) anti-FKBP51 (1:1000, ab2901, Abcam, Cambridge, UK), anti-CypA (1:2000, Catalog# ab126738, Abcam, Cambridge, UK), anti-tau H150 (1:200, Catalog# sc-5587, Santa Cruz, Dalla, TX, USA), anti-Pin1 (1:250, Catalog# sc-15340, Santa Cruz, Dalla, TX, USA), anti-Ahsa1 (1:1000, Catalog# 12841, Cell Signalling, Danvers, MA, USA), anti-Cdc37 (1:1000, Catalog# 4793, Cell Signalling, Danvers, MA, USA), anti-p23 (1:1000, Catalog#: NB300-576, Novus Biologicals; Biotechne, Littleton, CO, USA).

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

doi: 10.1038/srep12728

Figure Lengend Snippet: ( a ) Structure of the CDC37-nanoKAZ protein. CDC37 and nanoKAZ is fused in-frame with a glycine-serine linker. ( b ) Expression of CDC37-nanoKAZ protein in transfected 293T cells. CDC37-nanoKAZ was detected with antibodies against CDC37 or nanoKAZ. GAPDH was detected as an internal control. ( c ) Interactions of 3xFLAG-DYRK1A and 3xFLAG-DYRK4 with CDC37-nanoKAZ. Luminescence intensities of CDC37-nanoKAZ associated with 3xFLAG-DYRK1A and 3xFLAG-DYRK4 proteins, which were bound on 96-well plates coated with antibody against FLAG, are shown as fold-changes relative to that with EGFP (luminescence due to non-specific binding of CDC37-nanoKAZ). Bar graphs show means ± SD, ***p < 0.001 (n = 8). ( d ) Cartoon illustrating the structure of 3xFLAG-DYRK1A, in which the mutations used in this study and the CDC37 consensus binding site are shown. ( e ) Interactions of intact and mutated DYRK1A proteins with CDC37-nanoKAZ. Luminescence intensities of CDC37-nanoKAZ associated with 3xFLAG-DYRK1A proteins are shown as fold-changes relative to that with EGFP. Bar graphs show means ± SD, **p < 0.01 (n = 5).

Article Snippet: Rabbit monoclonal antibodies against CDC37 (D11A3), GSK3β (27C10), AKT1 (C73H10), MEK1 (30C8), SRC (32G6) and rabbit polyclonal antibodies against DYRK1A, DYRK1B, p42/p44 MAPK (ERK1/2), p38 MAPK, SAPK/JNK, p70S6K, CHK2, MARK3, CK1, CK2α, RAF1, FAK, FYN, c-ABL, and IKKα were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Transfection, Control, Binding Assay

Journal: Scientific Reports

Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

doi: 10.1038/srep12728

Figure Lengend Snippet: ( a-d ) 293T cells stably expressing CDC37-nanoKAZ were transiently transfected with an expression vector for 3xFLAG-DYRK1A (Y319F/Y321F) and then treated with the indicated concentrations of harmine ( a ), INDY ( b ), staurosporine ( c ), and ganetespib ( d ). Luminescence intensities are shown as fold-changes relative to that at 0 nM, normalized to the amount of 3xFLAG-DYRK1A (Y319F/Y321F) bound on a 96-well plate. Points are means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown.

Article Snippet: Rabbit monoclonal antibodies against CDC37 (D11A3), GSK3β (27C10), AKT1 (C73H10), MEK1 (30C8), SRC (32G6) and rabbit polyclonal antibodies against DYRK1A, DYRK1B, p42/p44 MAPK (ERK1/2), p38 MAPK, SAPK/JNK, p70S6K, CHK2, MARK3, CK1, CK2α, RAF1, FAK, FYN, c-ABL, and IKKα were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

doi: 10.1038/srep12728

Figure Lengend Snippet: ( a ) A total of 253 compounds from our original synthetic chemical library were examined. These compounds were used at 4 μM. Relative luminescence intensities are shown. Harmine was used as a positive control and is indicated by the black point. The red point indicates CaNDY. ( b ) Structure of CaNDY. ( c ) CaNDY antagonized the interaction between DYRK1A and CDC37 complex. 293T cells expressing CDC37-nanoKAZ were transiently transfected with an expression vector for 3xFLAG-DYRK1A (Y319F/Y321F) and then treated with the indicated concentrations of CaNDY. The points represent means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) CaNDY inhibited the catalytic activity of DYRK1A in an in vitro kinase assay. Recombinant DYRK1A was incubated with the substrate peptide DYRKtide-F in the presence of the indicated concentrations of small molecules. CaNDY, INDY, and staurosporine inhibited the kinase activity with IC 50 values of 7.9 nM, 122 nM, and 5.4 nM, respectively. Representative dose-response curves with Hill slopes are shown. ( e ) Double-reciprocal plots showing the competitive inhibition of ATP by CaNDY. DYRK1A kinase activity was measured at the indicated concentrations of CaNDY and ATP. Reciprocal velocity was plotted versus 1/[ATP]. K m = 14.1 μM and K i = 1.92 nM.

Article Snippet: Rabbit monoclonal antibodies against CDC37 (D11A3), GSK3β (27C10), AKT1 (C73H10), MEK1 (30C8), SRC (32G6) and rabbit polyclonal antibodies against DYRK1A, DYRK1B, p42/p44 MAPK (ERK1/2), p38 MAPK, SAPK/JNK, p70S6K, CHK2, MARK3, CK1, CK2α, RAF1, FAK, FYN, c-ABL, and IKKα were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Positive Control, Expressing, Transfection, Plasmid Preparation, Activity Assay, In Vitro, Kinase Assay, Recombinant, Incubation, Inhibition

Journal: Scientific Reports

Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

doi: 10.1038/srep12728

Figure Lengend Snippet: ( a ) Structures of PD0439, RD0440, and PD0442. ( b ) In vitro kinase assay using these small molecules, as performed in . ( c ) CDC37-nanoKAZ binding assay using these small molecules, as performed in .

Article Snippet: Rabbit monoclonal antibodies against CDC37 (D11A3), GSK3β (27C10), AKT1 (C73H10), MEK1 (30C8), SRC (32G6) and rabbit polyclonal antibodies against DYRK1A, DYRK1B, p42/p44 MAPK (ERK1/2), p38 MAPK, SAPK/JNK, p70S6K, CHK2, MARK3, CK1, CK2α, RAF1, FAK, FYN, c-ABL, and IKKα were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vitro, Kinase Assay, Binding Assay

Journal: Cancers

Article Title: On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation

doi: 10.3390/cancers11010097

Figure Lengend Snippet: List of antibodies.

Article Snippet: Rabbit , CDC37 (3618S) , WB 1:1000 , Cell Signaling.

Techniques:

Journal: Cell reports

Article Title: Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

doi: 10.1016/j.celrep.2023.112807

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Cdc37 , StressMarq Biosciences , Cat# SPC-142; RRID:AB_2570605.

Techniques: Virus, Recombinant, Proximity Ligation Assay, Software

Journal: eLife

Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin 2α -induced corpus luteum regression

doi: 10.7554/eLife.67409

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CDC37 (Rabbit monoclonal) , Abcam , Cat# ab108305; RRID: AB_10861724 , WB (1:1000) IF (1:100).

Techniques: Recombinant, Software