Journal: International Journal of Molecular Sciences

Article Title: Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors

doi: 10.3390/ijms22157872

Figure Lengend Snippet: Peroxisomal ether lipid and cholesterol synthesis in PMA. The mRNA expression by real-time PCR analysis of ( A ) Agps (** p = 0.0018), ( B ) Hmgcr (* p = 0.0235) and Pmvk (* p = 0.0478) are expressed lower while Gnpat (*** p = 0.0004) ( A ), Hmgcs, Idi (* p = 0.0155), Fdps (* p = 0.0257) and Sqs (** p = 0.0059) ( B ) are expressed higher in PMA than in healthy tissue. (Explanation of the notations: The number of asterices signifies the degree of significance of the p -value, while # means not significant.)

Article Snippet: Alkylglycerone-phosphate synthase (AGPS) , Mouse, monoclonal , 78 kDa , 1:1000 , 1:500 , Santa cruz, Cat no: sc-374201.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors

doi: 10.3390/ijms22157872

Figure Lengend Snippet: List of antibodies used for the detection of peroxisomal proteins in the parotid gland.

Article Snippet: Alkylglycerone-phosphate synthase (AGPS) , Mouse, monoclonal , 78 kDa , 1:1000 , 1:500 , Santa cruz, Cat no: sc-374201.

Techniques: Molecular Weight, Phospho-proteomics, Marker

Journal: International Journal of Molecular Sciences

Article Title: Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors

doi: 10.3390/ijms22157872

Figure Lengend Snippet: List of the human primers used for qPCR analyses.

Article Snippet: Alkylglycerone-phosphate synthase (AGPS) , Mouse, monoclonal , 78 kDa , 1:1000 , 1:500 , Santa cruz, Cat no: sc-374201.

Techniques:

Journal: Nature Cell Biology

Article Title: PEX39 facilitates the peroxisomal import of PTS2-containing proteins

doi: 10.1038/s41556-025-01711-z

Figure Lengend Snippet: a , Depiction of protein–protein interactions for Yjr012c and C6ORF226, per BioGRID and BioPlex , , respectively. The circles represent proteins and the arrows point from purified proteins to interactors. For Yjr012c, only interactions found in at least two independent studies were considered. For C6ORF226, the combined interactome from HCT116 and HEK293T cells is shown. b , Domain analysis and alignment of Yjr012c and C6ORF226. Probabilities of disorder were determined using IUPred2A . c , Quantitative proteomic analysis of Pex18 complexes that were affinity purified from soluble fractions of wild-type and Pex18-TPA-expressing yeast grown in oleic acid medium ( n = 3). The enrichment of proteins in Pex18 complexes and Q values were determined using the rank-sum method . Peroxins and PTS2-containing proteins are labelled and/or marked by black dots. The dashed lines indicate a Q -value threshold of 0.05 and a fold-enrichment of 64. d , Immunoblots of anti-FLAG immunoprecipitates prepared from HCT116 cells expressing the indicated proteins. HA denotes the detection of HA-tagged proteins. The dashed lines indicate where different lanes of the same membrane were brought together. For the PHYH, ACAA1 and AGPS blots, solid and open red arrowheads indicate mature and precursor forms of these proteins, respectively. An asterisk indicates a non-specific band. The control protein β-actin (ACTB) should be absent from immunoprecipitates. e , Immunoblots of anti-FLAG immunoprecipitates prepared from HEK293T cells expressing the indicated proteins. The control protein calnexin (CANX) should be absent from immunoprecipitates. The annotation of the immunoblots is otherwise the same as for d . f , Immunoblots of anti-FLAG immunoprecipitates prepared from HEK293T cells expressing FLAG-HA-eGFP or Hs PEX39-FLAG-HA. An asterisk indicates a non-specific band. g , Native PAGE and autoradiography of [ 35 S]H 6 PEX7 pre-incubated with the recombinant proteins GST- Hs PEX39, H 6 PHYH and H 6 PEX5(1–324), as indicated. The in-gel positions of PEX7 alone, lysate haemoglobin and the complexes PEX7– Hs PEX39 (hashtag), PEX7–PHYH– Hs PEX39 (ampersand) and PEX7–PEX5–PHYH– Hs PEX39 (dollar sign) are indicated. The autoradiograph and corresponding Ponceau S-stained membrane are shown. α, α-helix; IP, immunoprecipitate.

Article Snippet: Immunoblot analysis was performed with antibodies against ACAA1 (HPA007244; Sigma–Aldrich; diluted 1/2,000), ACOX1 (ab184032; Abcam; diluted 1/2,000), AGPS (described previously ; diluted 1/3,000), Hs PEX13 ( PAB22801 ; Abnova; diluted 1/2,000), PHYH (12858-1-AP; Proteintech; diluted 1/1,000) and TUBA4A (T6199; Sigma–Aldrich; diluted 1/2,000).

Techniques: Protein-Protein interactions, Purification, Affinity Purification, Expressing, Western Blot, Membrane, Control, Clear Native PAGE, Autoradiography, Incubation, Recombinant, Staining

Journal: Nature Cell Biology

Article Title: PEX39 facilitates the peroxisomal import of PTS2-containing proteins

doi: 10.1038/s41556-025-01711-z

Figure Lengend Snippet: a , Growth of the indicated yeast strains in oleic acid medium. pPEX39 is a plasmid containing Scpex39 under the control of its endogenous promoter. The data represent means ± s.d. ( n = 4). b , Immunoblots of cellular fractions from wild-type and Scpex39 Δ yeast. A post-nuclear supernatant (PNS) was prepared from cells grown in oleic acid medium and further separated into a cytosolic supernatant (S) and organellar pellet (OP). c , Quantification of the subcellular distribution of proteins using the band intensities of the immunoblots shown in b and Extended Data Fig. . For each protein, the summed intensities of the cytosolic supernatant and organelles were set to 100%. The data represent means ± s.e.m. ( n = 2 for Pex3 and n = 3 for the rest). Statistical significance was determined by unpaired, two-tailed t -test. d , Fluorescence microscopy of Pot1-mNeonGreen in control, Scpex39 Δ and pex7 Δ yeast grown in oleic acid medium. Peroxisomes were visualized with Pex3-mScarlet. Scale bar, 1 μm. e , Quantitative proteomic analysis of wild-type and Scpex39 Δ yeast ( n = 4). Proteins quantified in at least three biological replicates are shown, except for Sc Pex39 (quantified in one replicate). PTS2-containing proteins and additional peroxisomal proteins are indicated by yellow and black circles, respectively. Multiple-testing-adjusted P values were determined using the limma approach (moderated two-tailed t -test) and Benjamini–Hochberg method. The dashed line indicates an adjusted P value threshold of 0.05. f , Immunoblot analysis of control and HsPEX39 -KO CAKI-2 and NCI-H1792 cells. Precursor ACAA1 and AGPS were undetectable. CANX and citrate synthase (CS) were used as loading controls. g , Quantification of mature and precursor PHYH in HsPEX39 -KO cells using the band intensities of immunoblots prepared per f . Mature/precursor ratios were divided by the mean value of the corresponding control. The data represent means ± s.e.m. ( n = 3). Statistical significance was determined by unpaired, two-tailed t -test. h , Immunoblot analysis of PEX7 -KD ( PEX7 -knockdown) and HsPEX39 -KO CAKI-2 cells. The dashed lines indicate where different lanes of the same membrane were brought together. An asterisk indicates a non-specific band. In f and h , the solid and open red arrowheads indicate mature and precursor forms, respectively. ER, endoplasmic reticulum; l.e., long exposure; s.e., short exposure.

Article Snippet: Immunoblot analysis was performed with antibodies against ACAA1 (HPA007244; Sigma–Aldrich; diluted 1/2,000), ACOX1 (ab184032; Abcam; diluted 1/2,000), AGPS (described previously ; diluted 1/3,000), Hs PEX13 ( PAB22801 ; Abnova; diluted 1/2,000), PHYH (12858-1-AP; Proteintech; diluted 1/1,000) and TUBA4A (T6199; Sigma–Aldrich; diluted 1/2,000).

Techniques: Plasmid Preparation, Control, Western Blot, Two Tailed Test, Fluorescence, Microscopy, Knockdown, Membrane

Journal: Nature Cell Biology

Article Title: PEX39 facilitates the peroxisomal import of PTS2-containing proteins

doi: 10.1038/s41556-025-01711-z

Figure Lengend Snippet: a , Quantification of mature ACAA1, mature AGPS, PEX7, and PEX5 in HsPEX39 -knockout cells using band intensities of immunoblots prepared per Fig. . Loading-control intensities were used to normalize protein abundance across samples and the normalized abundances were then divided by the mean value of the corresponding control cells. The names of quantified proteins are shown on the bottom. Precursor forms of ACAA1 and AGPS were undetectable and thus not quantified. Data are mean ± standard error of the mean (SEM) (n = 3), and P values were calculated using unpaired, two-tailed t-tests. b , Immunoblot analysis of cells generated via lentiviral transduction of Cas9 and multiple independent sgRNAs against HsPEX39 (sg HsPEX39 _1-3) or a negative control sgRNA against the AAVS1 gene (sg AAVS1 ) in the CAKI-2 and NCI-H1792 cell lines. Note that the residual Hs PEX39 signal in these cells is because not all cells in the population will have complete loss of the protein. Solid and open red arrowheads indicate mature and precursor forms, respectively. Precursor forms of ACAA1 and AGPS were undetectable. CANX is a loading control. Short and long exposures are denoted s.e. and l.e., respectively. c , Immunoblots of whole-cell (Cell), cytosolic (Cyto), and organellar (Org) fractions prepared from the CAKI-2 cells expressing sg AAVS1 (negative control) or sg HsPEX39 _1 described in b . Solid and open red arrowheads indicate mature and precursor forms, respectively. The cellular fractions that RPS6KB1 and CANX are markers for are indicated in parentheses.

Article Snippet: Immunoblot analysis was performed with antibodies against ACAA1 (HPA007244; Sigma–Aldrich; diluted 1/2,000), ACOX1 (ab184032; Abcam; diluted 1/2,000), AGPS (described previously ; diluted 1/3,000), Hs PEX13 ( PAB22801 ; Abnova; diluted 1/2,000), PHYH (12858-1-AP; Proteintech; diluted 1/1,000) and TUBA4A (T6199; Sigma–Aldrich; diluted 1/2,000).

Techniques: Knock-Out, Western Blot, Control, Quantitative Proteomics, Two Tailed Test, Generated, Transduction, Negative Control, Expressing

Journal: Scientific Reports

Article Title: Arabinogalactan-proteins of Zostera marina L. contain unique glycan structures and provide insight into adaption processes to saline environments

doi: 10.1038/s41598-020-65135-5

Figure Lengend Snippet: Reactivity of Zostera marina AGPs and their partial hydrolyses (with oxalic acid = Ox; by reduction of uronic acids = UR) with antibodies directed against AGP glycans by ELISA. ( a ) KM1 antibody with E. purpurea AGP and A. sativa AGP as positive controls. ( b ) LM2 and LM6 antibodies. ( c ) oligosaccharides with strong binding affinity to KM1, LM2 and LM6, respectively . ( d ) KM4 antibody with A. sativa AGP as positive control. ( e ) JIM8 and JIM 13 antibodies.

Article Snippet: Studies on Medicago sativa leaves showed an increase of AGPs recognized by the JIM8 antibody in NaCl-treated plants .

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

doi: 10.1186/s13046-023-02920-w

Figure Lengend Snippet: TrkA promotes the ubiquitinated degradation of AGPS by MDM2 by modifying the phosphorylation of AGPS. a Western blot analysis of endogenous AGPS proteins in DU145 and 22Rv1 cells followed by treatment with different phosphatase inhibitors (Merestinib, 10 μM for 24 h; AG1295,10 μM for 2 h; AIM-100, 10 μM overnight; Dacomitinib, 2 μM for 24 h; Larotrectinib, 20 nM overnight). b Co-IP analysis of the phosphorylation levels of Flag-tagged AGPS in DU145 cells after being treated with MG132 (20 μM) for 8 h and Larotrectinib. c Western blot analysis of TrkA and p-TrkA expression in DU145, 22Rv1 and RWPE-1 cells. d Analysis of correlation between IHC staining of AGPS and p-TrkA proteins in different Gleason score PCa patient specimens. e Western blot analysis of AGPS protein expression in DU145 and 22Rv1 cells with or without Larotrectinib treatment. f Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with Larotrectinib. g Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with MG132 (20 μM) for 8 h or with Larotrectinib. h Western blot analysis of AGPS protein expression in DU145 cells with or without CHX (50 μg/mL) treatment for 0-, 4-, 8-, 12-, 16-, or 20- h. i Protein bands were quantified and normalized to the band intensity at the 0-h time point. * P < 0.05, ** P < 0.01, *** P < 0.001. j Co-IP analysis of binding of Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells after NTRK1 knockdown. k Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells after NTRK1 knockdown and treated with MG132 (20 μM) for 8 h

Article Snippet: These sections were dyed with diluted anti-AGPS and anti-p-TrkA antibody and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature.

Techniques: Phospho-proteomics, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunohistochemistry, Ubiquitin Proteomics, Binding Assay, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

doi: 10.1186/s13046-023-02920-w

Figure Lengend Snippet: Larotrectinib promotes the sensitivity of prostate cancer cells to ferroptosis by inhibiting the phosphorylation of TrkA. a Western blot analysis of AGPS protein expression in DU145 and 22Rv1 cells with or without ML210 (10 μM for 8 h) and Larotrectinib (20 nM, overnight) treatment. b The sensitive of ferroptosis in DU145 and 22Rv1 cells with or without ML210 and Larotrectinib treatment. ** P < 0.01, *** P < 0.001. c , d PMP70 staining in DU145 and 22Rv1 cells with or without ML210 and Larotrectinib treatment. ** P < 0.01, *** P < 0.001, **** P < 0.0001. e , f Pca cells were observed by TEM with or without ML210 and Larotrectinib treatment. ** P < 0.01, *** P < 0.001, **** P < 0.0001. g MDA level after treatment with ML210 and/or Larotrectinib in DU145 and 22Rv1 cells. * P < 0.05, ** P < 0.01. h CCK8 essay with the OD values in 6-wells plate after treatment with ML210 and/or Larotrectinib in DU145 and 22Rv1 cells. ** P < 0.01, *** P < 0.001, **** P < 0.0001. i Colony formation assay displayed the DU145 and 22Rv1 cell colony numbers after treatment with ML210 and/or Larotrectinib. j Organoid culture treated with ML210 and/or Larotrectinib from Pca patient tissues. k PCa cells were injected subcutaneously into the right flank of mice after treatment with ML210(50 mg/kg) and/or Larotrectinib (300 mg/kg) every other 24 h. Xenograft growth was measured every other day for 28 days. Tumors in each group at day 28 were harvested and photographed. l Tumor weight in different groups. Data represented as mean ± SD ( n = 5). ** P < 0.01, **** P < 0.0001. m Tumor volume at each time point. Data represented as mean ± SD ( n = 5). n.s., no significance; * P < 0.01, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: These sections were dyed with diluted anti-AGPS and anti-p-TrkA antibody and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature.

Techniques: Phospho-proteomics, Western Blot, Expressing, Staining, Colony Assay, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

doi: 10.1186/s13046-023-02920-w

Figure Lengend Snippet: A hypothetic model depicting the dual regulation mechanism of AGPS protein degradation by MDM2 and TrkA inhibitor. MDM2 ubiquitinates and promotes the degradation of AGPS, phosphorylation kinase TrkA aberrantly activated in prostate cancer and facilitates MDM2 ubiquitinate AGPS. TrkA inhibitor Larotrectinib could reactivate peroxisome pathway and sensitive prostate cancer cells to ML210. The combined administration of ML210 and Larotrectinib could significantly ablate the progression of prostate cancer.The model was created by Pro Procreate software and BioRender website ( www.biorender.com )

Article Snippet: These sections were dyed with diluted anti-AGPS and anti-p-TrkA antibody and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature.

Techniques: Phospho-proteomics, Software