anti cdc37  (Santa Cruz Biotechnology)

Journal: NAR Cancer

Article Title: Human DEAH-box helicase 8 regulates HSF1-mediated stress response and cancer-associated pre-mRNA splicing in tumour cells

doi: 10.1093/narcan/zcag008

Figure Lengend Snippet: DHX8 knockdown inhibits heat shock gene induction and alters HSF1 splicing in U2OS human osteosarcoma cells. Quantification of heat shock gene induction following HSP90 inhibition with 250 nM 17-AAG treatment. Bar graphs show the percentage induction of ( A ) HSPA1A mRNA or ( B ) HSPB1 mRNA. Percentage induction was calculated as a percentage of mean mRNA relative to the treatment with 250 nM 17-AAG and transfection with AllStars negative control siRNA condition. ( C ) Fold change in basal HSF1 mRNA levels following silencing of HSF1 or DHX8 (mean ± SEM, n ≥ 3) relative to negative control siRNA. ( D ) Immunoblot analysis of heat shock protein expression over time following siRNA-mediated knockdown of DHX8 or HSF1, following 250 nM 17-AAG treatment. GAPDH served as a loading control. AS = AllStars negative control siRNA, O1 = DHX8 siRNA 1, O3 = DHX8 siRNA 3, HSF = HSF1 siRNA. Immunoblots shown are exemplars of two independent repeats. Effects of HSF1 or DHX8 knockdown on transcript levels on non-heat shock-regulated control mRNAs: ( E ) NOXA , ( F ) MYC , and ( G ) CDC37 were quantified using RT-qPCR TaqMan (mean ± SD, n = 3). Impact of HSF1 or DHX8 knockdown for 72 h on RNA processing was assessed by RT-qPCR using TaqMan probes targeting intron 1 regions of ( H ) HSF1 or ( I ) NOXA (mean ± SEM, n ≥ 3, ** P < .01 determined using a two-tailed t -test comparing pre-mRNA levels between AllStars negative control and DHX8 siRNA conditions. ( J ) Data from RIP microarray analysis showing association of HSF1 or NOXA RNA with DHX8 (mean ± SEM, n = 5 *** P < .001 determined using a two-tailed t -test comparing pull-down with DHX8 antibody compared to the IgG control). Only significant differences are indicated on the plots.

Article Snippet: TaqMan assay primers were obtained from Applied Biosystems: HSPA1A (Hs00359163_s1), HSPB1 (Hs03044127_g1), HSF1 (Hs00232134_m1), un-spliced HSF1 (Hs03673241_cn), spliced NOXA (Hs00560402_m1), un-spliced NOXA (Hs00906409_cn), spliced MYC (Hs0153408_m1), CDC37 (Hs01003386_g1), and control RPLP0 (4326314E).

Techniques: Knockdown, Inhibition, Transfection, Negative Control, Western Blot, Expressing, Control, Quantitative RT-PCR, Two Tailed Test, Microarray

Journal: The Journal of Biological Chemistry

Article Title: Targeting the protein–protein interaction between the CDC37 cochaperone and client kinases by an allosteric RAF dimer breaker

doi: 10.1016/j.jbc.2025.111018

Figure Lengend Snippet: Braftide disrupts the interaction between the client RAF kinase and the CDC37 cochaperone. A – D, BRAF-FLAG ( A and B )/MBP-CRAF-FLAG ( C and D ) exogenously expressed in HEK293 cells in the absence and presence of 50 μM braftide treatment (4 h). Representative immunoblot of immunoprecipitated (IP) B/CRAF-FLAG for the coimmunoprecipitated (co-IP) CDC37 and HSP90 chaperone complex. B and D, densitometry analysis of CDC37 and HSP90 normalized to immunoprecipitated BRAF ( B )/CRAF ( D ) across three biological replicates. E – H, CDC37-FLAG coexpressed with BRAF-V5 ( E and F ) or CRAF-HA ( G and H ). Representative blot of three biological replicates of immunoprecipitated CDC37-FLAG for co-IP BRAF-V5 ( E ) or CRAF-HA ( G ) and HSP90 in the absence and presence of braftide treatment (25 μM) in HEK293 cells. I, schematic illustrating NanoBiT luminescence assay. J and L, NanoBiT assay in the absence and presence of braftide (25 μM, 4 h) in HEK293 cells expressing NanoBiT constructs of BRAF SmBiT –CDC37 LgBiT ( J ) and CRAF SmBiT –CDC37 LgBiT ( L ). K and M, representative immunoblot of HEK239 cells expressing BRAF SmBiT –CDC37 LgBiT ( K ) and CRAF SmBiT –CDC37 LgBiT ( M ) in the absence and presence of braftide. K – M, densitometry analysis of CDC37 LgBiT and BRAF SmBiT ( K ) or CRAF SmBIT ( M ) normalized to no treatment control across three biological replicates. At least three independent biological replicates were performed for each experiment. HEK293, human embryonic kidney 293 cell line; HSP90, heat shock protein 90; LgBiT, large BiT; SmBiT, small BiT.

Article Snippet: Cell Signaling , 4222 , CDC37.

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Luminescence Assay, Expressing, Construct, Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting the protein–protein interaction between the CDC37 cochaperone and client kinases by an allosteric RAF dimer breaker

doi: 10.1016/j.jbc.2025.111018

Figure Lengend Snippet: Braftide binds to CDC37 and RAF kinases and induces proteasomal degradation of the client kinase via the ubiquitin–proteasome pathway. A and B, cartoon schematic of photo-activated crosslinking (XLINK) strategy of biotinylated (BTN)–braftide ( B ) with BRAF. Crosslinked BTN–braftide complexes are coimmunoprecipitated via streptavidin magnetic resin for further analysis. C, representative immunoblot of overexpressed BRAF-V5 and CDC37-FLAG in HEK293 cells in the absence and presence of 10 μM BTN–braftide and then immunoprecipitated and probed for individual proteins. D and E, representative immunoblot ( D ) and densitometry ( E ) of overexpressed MBP-CRAF-FLAG immunoprecipitated and probed for ubiquitination in HEK293 cells in the absence and presence of 50 μM braftide. At least three independent biological replicates were performed for each experiment. F and G, braftide-mediated CDC37 disruption triggers apoptosis in HEK293 ( F ) cells and HCT116 ( G ) cells treated with indicated concentrations of braftide for 4 h. Cells were stained with Annexin V, an apoptosis marker, and sorted via flow cytometry (n = 3). HEK293, human embryonic kidney 293 cell line.

Article Snippet: Cell Signaling , 4222 , CDC37.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Disruption, Staining, Marker, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Targeting the protein–protein interaction between the CDC37 cochaperone and client kinases by an allosteric RAF dimer breaker

doi: 10.1016/j.jbc.2025.111018

Figure Lengend Snippet: The HxNI motif is a key interaction site between CDC37 and client kinases. A, overlay of the HxNI motifs of CDC37 (Protein Data Bank [PDB] code: 7ZR0 ) in yellow and BRAF KD (PDB code: 4E26 ) in magenta . B and E, BRAF-V5 ( B and C )/CRAF-HA ( D and E ) coexpressed with CDC37-FLAG WT or CDC37-FLAG HxNI→AxAA in HEK293 cells. Representative blot of three biological replicates of immunoprecipitated CDC37-FLAG for coimmunoprecipitated (co-IP) BRAF-V5 ( B ) or CRAF-HA ( D ) and HSP90. C and E, densitometry analysis of immunoprecipitated BRAF ( C )/CRAF ( E ) across three biological replicates. F, NanoBiT assay with CDC37-FLAG WT or CDC37-FLAG HxNI→AxAA in HEK293 cells expressing NanoBiT constructs of BRAF SmBiT –CDC37 LgBiT . G, interactions between CDC37 and braftide identified by MD simulation are shown in one overview ( middle ) and two zoomed-in views depicting molecular details of hydrogen bonds ( left ) and hydrophobic interactions ( right ). H and I, HCT116 cells treated with braftide ( H ) or braftide R2H mutant ( I ) at the indicated concentrations for 4 h. Cells were harvested, lysed, and immunoblotted for MAPK proteins (BRAF, CRAF, MEK, phosphorylated MEK [pMEK], ERK, and pERK). Densitometry analysis of pMEK and pERK normalized to 0 μM braftide ( H ) or braftide R2H ( I ). At least three independent biological replicates were performed for each experiment. ERK, extracellular signal–regulated kinase; HEK293, human embryonic kidney 293 cell line; HSP90, heat shock protein 90; MD, molecular dynamics.

Article Snippet: Cell Signaling , 4222 , CDC37.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Construct, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Targeting the protein–protein interaction between the CDC37 cochaperone and client kinases by an allosteric RAF dimer breaker

doi: 10.1016/j.jbc.2025.111018

Figure Lengend Snippet: Braftide disrupts RAF dimers and the CDC37–RAF client kinase complex. A, braftide binds to the dimer interface of RAF to dissociate RAF dimers. B, braftide binds to CDC37 to disrupt the cochaperone CDC37 with client kinases. Braftide treatment increases the ubiquitination of RAF, which leads to proteasomal-mediated degradation.

Article Snippet: Cell Signaling , 4222 , CDC37.

Techniques: Ubiquitin Proteomics