Journal: Human Molecular Genetics

Article Title: PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

doi: 10.1093/hmg/ddae051

Figure Lengend Snippet: Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in liver. (A and B) Western blot analysis of PAH, ubiquitin and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in liver samples from 12-week-old mice. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Statistical analysis performed by unpaired t test ( * P < 0.05, ** P < 0.01, ( *** P < 0.001, **** P < 0.0001, ns, not significant). Data are presented as mean ± SEM.

Article Snippet: For the analysis of total proteins , membranes were blocked for 1 h with 5% nonfat milk, in 0.1% TBS-tween and incubated overnight with the corresponding primary antibody: PAH (1:1000, Santa Cruz Biotechnology, sc-271 258), Ubiquitin (1:1000, Santa Cruz Biotechnology, sc-8017), Hsp70 (1:1000, Novus Biological, NB110-61582), DNAJC12 (1/1000, Abcam, ab167425), LC3B (1:1000, Cell Signaling Technology, #2775), LAMP1 (1:1000, Cell Signaling Technology, #3243) and p62/SQSTM1 (1:5000, Novus biologicals, H00008878-M01).

Techniques: Variant Assay, Western Blot, Ubiquitin Proteomics, Control

Journal: Human Molecular Genetics

Article Title: PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

doi: 10.1093/hmg/ddae051

Figure Lengend Snippet: Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in HepG2 cells. (A and B) Representative western blot analysis of PAH and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in HepG2 cells. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Data are presented as mean ± SD of two independent experiment. (D) PAH levels detected by western blot in HepG2 cells 72 h after transfection with 2 μg of the plasmid coding for DNAJC12. GAPDH was used as loading controls.

Article Snippet: For the analysis of total proteins , membranes were blocked for 1 h with 5% nonfat milk, in 0.1% TBS-tween and incubated overnight with the corresponding primary antibody: PAH (1:1000, Santa Cruz Biotechnology, sc-271 258), Ubiquitin (1:1000, Santa Cruz Biotechnology, sc-8017), Hsp70 (1:1000, Novus Biological, NB110-61582), DNAJC12 (1/1000, Abcam, ab167425), LC3B (1:1000, Cell Signaling Technology, #2775), LAMP1 (1:1000, Cell Signaling Technology, #3243) and p62/SQSTM1 (1:5000, Novus biologicals, H00008878-M01).

Techniques: Variant Assay, Western Blot, Control, Transfection, Plasmid Preparation

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

Journal: Cell death and differentiation

Article Title: Combined absence of TRP53 target genes ZMAT3, PUMA and p21 cause a high incidence of cancer in mice.

doi: 10.1038/s41418-023-01250-w

Figure Lengend Snippet: Fig. 3 The combined absence of ZMAT3 and p21 does not accelerate γ-radiation induced thymic lymphoma development. A Kaplan- Meier curves showing percentages of tumour-free mice of the indicated genotypes after exposure to four weekly doses of γ-radiation (1.5 Gy for each dose). Differences in thymic lymphoma incidence between wt and p21−/−Zmat3−/−were not statistically significant. P value determined by log-rank (Mantel-Cox) test p = 0.5. B Immunophenotyping of γ-radiation-induced thymic lymphomas arising in mice of the indicated genotypes, as assessed by cell surface marker staining and flow cytometric analysis of tumour cells from the thymus. Data are presented as the frequency of the indicated phenotypes for each genotype. Double negative CD4-CD8- (DN) or double positive CD4+CD8+ (DP) lymphoma cells found in the thymus that show a trend towards the CD8 T cell lineage (DP/CD8+ or DN CD8+, respectively). N number of mice. C Thymus weights from sick mice of the indicated genotypes. Significant differences were observed in spleen weights between the sick p21−/−Zmat3−/−and sick wt γ-irradiated mice. p21−/−Zmat3−/−(N = 12), p21−/−(N = 7), Zmat3−/−(N = 7) and wt (N = 5). Mean ± SEM, Unpaired Students t-test **p = 0.0065. D Western blot analysis of p19ARF, TRP53 and HSP70 (loading control) in thymic lymphomas of the indicated genotypes. Red asterisk indicates samples with dysregulated TRP53 pathway. The Trp53 mutant Eμ-Myc lymphoma cell line EMRK1172 [51] was used as a control for p19ARF and mutant TRP53 protein overexpression. Protein size standards in kilodaltons (kDa) are indicated. The numbers represent the identification of individual mice in the colony. E Trp53 exons 4-11 were sequenced from tumours (N = 26, Table S4). Table summary of mutations found, listed by amino acid change, Trp53 knock out (KO) or Trp53 wild-type (WT) alleles. F Summary of the TRP53 status in γ-radiation-induced thymic lymphomas arising in mice of the indicated genotypes as assessed by Western blot analysis and/or Trp53 exon sequencing (see also Table S4). Inconclusive refers to lymphomas where TRP53 pathway dysregulation was seen by only Western blot analysis or exon sequencing but not in both assays.

Article Snippet: The following antibodies were used for probing; mouse TRP53 (clone CM5, Novocastra), p19/ARF (clone 5.C3.1, Rockland) and HSP70 (clone N6, gift from Dr. R Anderson, Olivia Newton-John Cancer Research Institute, Melbourne, VIC, Australia), the latter used as a control for protein loading.

Techniques: Marker, Staining, Irradiation, Western Blot, Control, Mutagenesis, Over Expression, Knock-Out, Sequencing

Journal: bioRxiv

Article Title: Exacerbation of mitochondrial fission in human CD34 + cells halts erythropoiesis and hemoglobin biosynthesis

doi: 10.1101/2020.07.31.230961

Figure Lengend Snippet: Total protein lysates from FIS1 OX and pWPI control cells at D8, D10, D13 and D16 of erythroid differentiation were examined by Western blot. (A) Protein levels of Procaspase 9 and cleaved active forms P37 and P35. This is for intrinsic apoptosis. HSP70 were used as housekeeping control. Band densitometry for p37, as a percentage of total caspase 9, was plotted. Values are +/− SEM. n =3. Statistical analysis, ANOVA followed by Bonferroni post hoc test. (B) Protein levels of Procaspase 8 and cleaved active forms P42/P43. This is for extrinsic apoptosis. HSP70 were used as housekeeping control. (C) Protein levels of Procaspase 3 and cleaved active form P17. This is a general apoptotic effector. HSP70 were used as housekeeping control. Band densitometry for p17, as a percentage of total caspase 3, was plotted. Values are +/− SEM. n =3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (*** p <0.001).

Article Snippet: Total proteins were run on SDS-PAGE, transferred to PVDF membranes and blotted with the antibodies against FIS1 (ab71498, Abcam), MFN1 (ab104274,Abcam), ß-Actin (ab8227, Abcam), Caspase-3 (9665, Cell Signaling), Caspase-8 (sc-5263, Santa Cruz), Caspase-9 (sc-8355, Santa Cruz), HSP70 (TA309356, Origene) and Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam).

Techniques: Control, Western Blot