Journal: American journal of hematology

Article Title: Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity.

doi: 10.1002/ajh.20885

Figure Lengend Snippet: Figure 6. Comparison of normalized Igb mRNA levels and ZAP70 expression in CLL patients. As in Fig. 4, the normalized Igb mRNA levels are shown for individual patients depicted as dots. The individuals are grouped by ZAP70 expression status. Horizontal lines represent the mean in each group. The significance of the pair-wise comparison is indicated by the P value below the graph.

Article Snippet: Immunohistochemistry was performed on five micron paraffin block sections on the DakoCytomation Autostainer (DakoCytomation Corporation, Carpinteria, CA) using the following antibodies: CD3 (Rabbit Polyclonal, 1:60, Neomarkers, Fremont, CA), CD5 (Mouse Monoclonal, clone 4C7, 1:25, Neomarkers), CD20 (Mouse Monoclonal, clone L26, 1:150, DakoCytomation), CD23 (Mouse Monoclonal, clone MHM6, Predilute, DakoCytomation), CD79a (Mouse Monoclonal, clone JCB117, 1:25, DakoCytomation) and ZAP70 (Mouse Monoclonal, clone 2F3.2, 1:80, Upstate Cell Signaling Solutions, Lake Placid, NY).

Techniques: Comparison, Expressing

Journal: American journal of hematology

Article Title: Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity.

doi: 10.1002/ajh.20885

Figure Lengend Snippet: Figure 7. Comparison of SHM status, Igb mRNA expression, and ZAP70 expression in CLL with levels of clinical sever- ity. A. Igb mRNA levels. Patients were stratified into low, intermediate, or high disease severity groups as described in materials and methods. The bars represent the percentage of patients in each clinical category that had high Igb mRNA levels. (n = 33) B. IgVH gene mutation. As in A. (n = 33) C. ZAP70 expression. As in A. (n = 29).

Article Snippet: Immunohistochemistry was performed on five micron paraffin block sections on the DakoCytomation Autostainer (DakoCytomation Corporation, Carpinteria, CA) using the following antibodies: CD3 (Rabbit Polyclonal, 1:60, Neomarkers, Fremont, CA), CD5 (Mouse Monoclonal, clone 4C7, 1:25, Neomarkers), CD20 (Mouse Monoclonal, clone L26, 1:150, DakoCytomation), CD23 (Mouse Monoclonal, clone MHM6, Predilute, DakoCytomation), CD79a (Mouse Monoclonal, clone JCB117, 1:25, DakoCytomation) and ZAP70 (Mouse Monoclonal, clone 2F3.2, 1:80, Upstate Cell Signaling Solutions, Lake Placid, NY).

Techniques: Comparison, Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis

doi: 10.1016/j.jbc.2025.110914

Figure Lengend Snippet: FKBP4 prevents CCT8 aggregation. A , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT1 and CCT8 proteins within cells was assessed using a filter trap assay. Cell lysates were filtered through a nitrocellulose membrane, and the retained proteins on the membrane were detected using specific antibodies against CCT1 and CCT8. B , quantitative analysis of CCT1 and CCT8 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. C , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT8, CCT1, CDK2, α-tubulin, and GAPDH within the cells. D , quantitative analysis of CCT8 and CCT1 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. E , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT2 and CCT3 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). F , quantitative analysis of CCT2 and CCT3 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. G , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT2, CCT3, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). H , quantitative analysis of CCT2 and CCT3 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. I , shFKBP4 HCT116 cells were harvested after 3 days of puromycin selection. The cell lysates were biochemically fractionated into Triton-soluble and -insoluble fractions as described in “ ”. The FKBP4 expression levels and Triton-soluble and -insoluble fractions of CCT8 were analyzed by western blotting. The GAPDH was used as a loading control. J , The ratio of the CCT8 Triton-insoluble form in ( I ). K , The primary structures of FKBP4 and FKBP5. L , after 48 h of transfection, vector, FLAG-tagged FKBP4 or FLAG-tagged FKBP5 expressing HEK293T cell lysates were precipitated by anti-FLAG antibodies, and the products were detected for the co-purification of the endogenous proteins. M , comparison of the interaction between Hsp90 and FLAG-tagged FKBP4 or FLAG-tagged FKBP5 in ( L ). N , following the knockdown of FKBP5 in HCT116 cells for 72 h, the aggregation of CCT8 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). O , quantitative analysis of the protein aggregation of CCT8, normalized to GAPDH, in shFKBP5 HCT116 cells. P , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP5, CCT8, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). Q , quantitative analysis of CCT8 protein expression, normalized to GAPDH, in shFKBP5 HCT116 cells. R , shFKBP5 HCT116 cells were harvested after puromycin selection for 3 days. The experimental procedure mirrored that of ( I ). S , the ratio of the CCT8 Triton-insoluble form in ( R ). T , HCT116 cells were treated with 17-AAG (20 μM) or ganetespib (2 μM) for 24 h, and CCT8 aggregation was assessed by filter trap assay. U , quantitative analysis of CCT8 protein aggregation with GAPDH serving as an internal control in ( T ). V , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of HSPA1A, CCT8, HSP90, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). W , quantitative analysis of CCT8 protein expression was normalized with GAPDH in ( V ). All data were analyzed with an unpaired two-tailed Student's t test. Each dataset is expressed as mean ± SD for n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies were used to detect FKBP4 (1:1000, ab97306, Abcam), GAPDH (1:1000, GTX100118, GeneTex, USA), E-cadherin (1:1000, 610182, BD), FASN (1:1000, GTX109833, GeneTex), FLNA (1:1000, GTX112939, GeneTex), TALIN-1 (1:500, GTX102215, GeneTex), MRE11 (1:1000, PC388, Calbiochem), DDX3 (1:1000, GTX110614, GeneTex), TCP1 theta (CCT8) (1:1000, GTX105725, GeneTex), CDK2 (1:500, 2546, Cell Signaling), CCT1 (1:250, sc-53454, Santa-cruz Biotechnology), HSP90 (1:1000, sc-13119, Santa-cruz Biotechnology), HSPA1A (1:1000, sc-66048, Santa-cruz Biotechnology), FKBP5 (1:1000, GTX113438, GeneTex), α-tubulin (1:1000, MCA78G, Bio-Rad Laboratories Inc), FLAG (1:2000, F3165, Sigma-Aldrich), c-Myc (1:1000, #11667203001, Roche), CCT2 (1:500, sc-374152, Santa-cruz Biotechnology), CCT3 (1:500, sc-271336, Santa-cruz Biotechnology), HA (1:5000, MMS-101R, Covance), LexA (1:1000, sc-7544, Santa-cruz Biotechnology), and Pgk1 (1:10000, 459250, Invitrogen).

Techniques: Knockdown, TRAP Assay, Membrane, Western Blot, Expressing, Selection, Control, Transfection, Plasmid Preparation, Copurification, Comparison, Two Tailed Test