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: Nature communications

Article Title: HSF1 phosphorylation establishes an active chromatin state via the TRRAP-TIP60 complex and promotes tumorigenesis.

doi: 10.1038/s41467-022-32034-4

Figure Lengend Snippet: Fig. 1 | Identification of the co-activator TRRAP that interacts with HSF1. a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty- one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of HSP72 mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with

Article Snippet: The following antibodies were used: rabbit antibodies for mHSF1 (antimHSF1n, Nakai lab, 1/1000), hTRRAP (anti-hTRRAP-2, Nakai lab, 1/ 1000), hTRIM24 (anti-hTRIM24-3, Nakai lab, 1/1000), hTRIM33 (antihTRIM33-2, Nakai lab, 1/1000), hPLK1 (anti-hPLK1-1, Nakai lab, 1/1000), GST (anti-GST, Nakai lab, 1/1000),mHSP110 (anti-mHSP110a, Nakai lab, 1/1000), hHSP90 (anti-hHSF90d, Nakai lab, 1/1000), hHSP40 (antihHSP40a,Nakai lab, 1/1000)15, p300 (SantaCruz, sc-585, 1/1000), p400 (Novus Biologicals, NB200-210, 1/1000), H2B (Abcam, ab1790, 1/ 1000), ubiquityl-histone H2B (Lys120) (Cell Signaling, 5546, 1/1000), RNF40 (GeneTex, GTX115295, 1/1000), histone H3 (Abcam, ab1791, 1/ 1000), H3 (acetyl K18) (Abcam, ab1191, 1/1000), Lamin B1 (Abcam, Ab16048, 1/1000), HSF1 (phosphor S326) (Abcam, ab115702, 1/1000) and HSF1 (phosphoS419) (anti-HSF1 phospho-S419b, Nakai lab, 1/ 1000), mouse antibodies for PLK1 (Santa Cruz, sc-17783, 1/1000), HSP72 (Santa Cruz, sc-24, 1/1000), ubiquitin (Santa Cruz, sc-8017, 1/ 1000), CSNK1A1 (Santa Cruz, sc-74582, 1/1000), NEK7 (Santa Cruz, sc393539, 1/1000), RNF20 (Santa Cruz, sc-517358, 1/1000), β-actin (Milipore Sigma, A5441, 1/1000), and GFP (Nacalai Tesque, GF200, 1/ 1000), rat antibody for HA (Roche, ROAHAHA, 1/1000), and goat Nature Communications | (2022) 13:4355 12 antibody for TIP60 (Santa Cruz, N-17, 1/200).

Techniques: Immunoprecipitation, Western Blot, Expressing, Control, ChIP-sequencing, Binding Assay

Journal: Acta pharmacologica Sinica

Article Title: Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis.

doi: 10.1038/aps.2011.147

Figure Lengend Snippet: Figure 6. Effect of M3 on the expression of Hsp70 at different time. (A) Analysis of Hsp70 protein expression in PC12 cells by western blotting at different time after exposure to M3 or HupA and the quantification of protein levels compare to the 0 h treating group by density scanning.

Article Snippet: The anti-Hsp70 antibody and antimouse secondary antibody were bought from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis.

doi: 10.1038/aps.2011.147

Figure Lengend Snippet: Figure 7. Quercetin inhibited the protective effect of M3 against SNP- induced apoptosis in PC12 cells. PC12 cells were pre-treated with 10 μmol/L HupA, M3, or 10 μmol/L quercetin for 1 h then exposed to 200 μmol/L SNP for the next 24 h. (A) Effect of quercetin on the cell viability in PC12 cells by MTT method. The percentage of cell viability was presented as mean±standard deviation for six replicates. (B) Effect of quercetin on the ratio of Caspase-3 and Hsp70 in PC12 cells. The percentage of density was presented as mean±standard deviation for three individual experiments. cP<0.01 vs control group. eP<0.05 vs SNP group. hP<0.05 vs M3 pretreated group.

Article Snippet: The anti-Hsp70 antibody and antimouse secondary antibody were bought from Santa Cruz Biotechnology.

Techniques: Standard Deviation, Control

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

Journal: International Journal of Molecular Sciences

Article Title: Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1

doi: 10.3390/ijms17091419

Figure Lengend Snippet: Immune system processing related differential proteins (21proteins).

Article Snippet: The target proteins were detected with the antibodies of EDIL3 (EGF-like repeat and discoidin I-like domain-containing protein 3; cat# 12580-1-AP), STAT1 (signal transducer and activator of transcription 1, cat# 10144-2-AP), PRDX1 (peroxiredoxin-1, cat# 15816-1-AP), HSPA4 (heat shock 70 kDa protein 4, cat#21206-1-AP), GSTO1 (glutathione S -transferase ω-1, cat# 15124-1-AP), and β-Actin (14395-1-AP) (Proteintech, Wuhan, China), followed by incubation with a secondary antibody conjugated with HRP.

Techniques: Activity Assay, Immunopeptidomics, Virus, Infection, Control, Cell Differentiation

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