Journal: Communications Biology

Article Title: Glutathione S-transferase Mu 2 inhibits hepatic steatosis via ASK1 suppression

doi: 10.1038/s42003-022-03251-w

Figure Lengend Snippet: a – c PCYT1A expression level was down-regulated by GSTM2 KD and up-regulated by GSTM2 OE; * p < 0.05, ** p < 0.01. d PCYT1A KD induced LD expansion. e , f GSTM2 affected recruitment of GPAT4 and DGAT2 on LD surface (in the presence of oleic acid medium), bar, 10 μm, 1 μm (inlay). g Expression level of GPAT4 and DGAT2 was regulated by treatment of palmitic acid (ASK1 activator) and GS-4997 (ASK1 activity inhibitor). h Recruitment of GPAT4 on LD surface was regulated by palmitic acid or GS-4997 treatment, bar, 10 μm, 1 μm (inlay). i , j qPCR detection of PCYTA1 expression with PA or GS-4997 treatment respectively.

Article Snippet: The following rabbit polyclonal antibodies were used: anti-GSTM2 (#ab125,102, Abcam, Cambridge, UK, 1:1000), anti-GSTM2 (#A13,496, ABclonal, Wuhan, China, 1:2000), anti-GAPDH (#CSB-PA00025A0Rb Flarebio Biotech LLC, College Park, MD, USA, 1:1000), anti-CIDEC (#D222114, Sangon Biotech, Shanghai, China, 1:1000), anti-CIDEC (#12287-1-AP, Proteintech, Rocky Hill, NJ, USA, 1:1000), anti-ADRP/perilipin2 (#15294-1-AP, Proteintech), anti-DGAT2 (#bs-12,998R, BIOSS, Beijing, China), anti-DGAT2 (#A13,891, ABclonal, Wuhan, China, 1:2000), anti-GPAT4 (#bs-15,587R, BIOSS), anti-SREBP1 (#14,088-1-AP, Proteintech), and anti-PCYT1A (#bs-11306R, BIOSS), anti-PCYT1A (#A5935, ABclonal, Wuhan, China, 1:1000).

Techniques: Expressing, Activity Assay

Journal: Communications Biology

Article Title: Glutathione S-transferase Mu 2 inhibits hepatic steatosis via ASK1 suppression

doi: 10.1038/s42003-022-03251-w

Figure Lengend Snippet: GSTM2 was highly up-regulated in hepatic steatosis tissues. As the member of phase II drug metabolizing enzymes, GSTM2 could detoxify the toxicity of metabolic products such as ROS. ROS could enhance the inflammation and fibrosis progression. Furthermore, GSTM2 interacted with ASK1 directly and suppressed ASK1 phosphorylation. Therefore, GSTM2 down-regulated expression of SREBP and perilipins to inhibit the LD accumulation. ASK1 signaling also regulated the expression level of DGAT2, GPAT4 and PCYT1A and the recruitment of DGAT2 and GPAT4 on LD surface to modulate the LD growth. In conclusion, GSTM2 protects against the hepatic steatosis of NAFLD via its antioxidant and suppressing ASK1-p38/JNK signaling.

Article Snippet: The following rabbit polyclonal antibodies were used: anti-GSTM2 (#ab125,102, Abcam, Cambridge, UK, 1:1000), anti-GSTM2 (#A13,496, ABclonal, Wuhan, China, 1:2000), anti-GAPDH (#CSB-PA00025A0Rb Flarebio Biotech LLC, College Park, MD, USA, 1:1000), anti-CIDEC (#D222114, Sangon Biotech, Shanghai, China, 1:1000), anti-CIDEC (#12287-1-AP, Proteintech, Rocky Hill, NJ, USA, 1:1000), anti-ADRP/perilipin2 (#15294-1-AP, Proteintech), anti-DGAT2 (#bs-12,998R, BIOSS, Beijing, China), anti-DGAT2 (#A13,891, ABclonal, Wuhan, China, 1:2000), anti-GPAT4 (#bs-15,587R, BIOSS), anti-SREBP1 (#14,088-1-AP, Proteintech), and anti-PCYT1A (#bs-11306R, BIOSS), anti-PCYT1A (#A5935, ABclonal, Wuhan, China, 1:1000).

Techniques: Expressing

Journal: Bone & Joint Research

Article Title: Unravelling lumbar disc herniation severity beyond MRI

doi: 10.1302/2046-3758.145.BJR-2024-0071.R1

Figure Lengend Snippet: The protein and messenger RNA (mRNA) expression level of lysophospholipid acyltransferase 3 (LPCAT3) and PCYT1A in the severe and mild groups. a) to c) Representative western blot strips of LPCAT3 and PCYT1A in the severe and mild groups with quantitative analysis (n = 3). d) and e) Quantitative real-time polymerase chain reaction (qRT-PCR) results of LPCAT3 and PCYT1A in the lumbar disc herniation (LDH) patients with mild and severe symptoms with relative quantitative analysis (n = 3). f) and g) Enzyme-linked immunosorbent assay (ELISA) results of LPCAT3 and PCYT1A with quantitative analysis (n = 3). Independent-samples t -test was used for comparison: *p < 0.05, **p < 0.01, ***p < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Primary antibodies, including anti-lysophospholipid acyltransferase 3 (LPCAT3) antibody (1:500, 67882-1-Ig; Proteintech, USA), anti-phosphate cytidylyltransferase 1, choline, alpha (PCYT1A) antibody (1:500, NBP1-84366; NOVUS, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,000, 10494-1-AP; Proteintech), were incubated overnight.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: BMC biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: Figure 2 The identification of the 8F1 cross-reactive protein with protein microarray chip and Western blot confirmation. A. Protein microarray hybridization with 8F1. The OriGene overexpression protein microarray chip was immunostained with the most commonly used 8F1 monoclonal anti-ERCC1 antibody. The positive reactive proteins are highlighted with red arrows. These data show that 8F1 recognizes not only its specific target (two ERCC1 transcript variants), but also another unrelated nuclear membrane protein PCYT1A. A number of internal controls were also labeled on the panel. B. Western Blot analysis. Seven OriGene VERIFY™overexpression lysate antigen standards (Lane 1 to 7) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (Upper panel). The recombinant protein expression levels within the lysates were analyzed with anti-DDK antibody (Lower panel). Lanes 1 to 7 are loaded with samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and PCYT1A (NM_005017).

Article Snippet: Primers used for ERCC1 mRNA detection were HP229725 with 50- GCTGGCTAAGATGTGTATCCTGG-30 (forward) and 5-ATCAGGAGGTCCGCTGGTTTCT-3 (reverse), for PCYT1A were HP208235 with 50-GTTCCTTCCAAA GTGCAGCGCT-30 (forward) and 5-AGGAGTTCCTC TGCTGGCTTCT-3, for β-actin were HP204660 with 50-CAGCCATGTACGTTGCTATCCAGG-30 (forward) and 5-AGGTCCAGACGCAGGATGGCATG-3 (OriGene Technologies Inc.).

Techniques: Microarray, Western Blot, Hybridization, Over Expression, Membrane, Labeling, SDS Page, Recombinant, Expressing

Journal: BMC biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: Figure 3 8F1 immunoblot analyses with purified proteins and antigen absorption test. A. Coomassie staining of purified ERCC1 and PCYT1A proteins. 1ug of affinity purified recombinant ERCC1 and PCYT1A proteins were fractionated on the SDS-PAGE gel and then commassie-stained. B. Immunoblot analysis with 8F1 anti-ERCC1 mAb.0.5ug of purified ERCC1 (Lane 2) and PCYT1A (Lane 4) were loaded on an SDS-PAGE gel and then immunobloted with 8F1 antibody. Empty vector transfected HEK293T cell lysates (Lanes 1 and 3) were used as a negative control. C. Antigen absorption test. Overexpression lysates for PCYT1A (Lane1), ERCC1 (Lane 3), and empty vector transfected control (Lanes 2 and 4) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (upper panel) or 8F1 pre-depleted with purified PCYT1A protein (lower panels).

Article Snippet: Primers used for ERCC1 mRNA detection were HP229725 with 50- GCTGGCTAAGATGTGTATCCTGG-30 (forward) and 5-ATCAGGAGGTCCGCTGGTTTCT-3 (reverse), for PCYT1A were HP208235 with 50-GTTCCTTCCAAA GTGCAGCGCT-30 (forward) and 5-AGGAGTTCCTC TGCTGGCTTCT-3, for β-actin were HP204660 with 50-CAGCCATGTACGTTGCTATCCAGG-30 (forward) and 5-AGGTCCAGACGCAGGATGGCATG-3 (OriGene Technologies Inc.).

Techniques: Western Blot, Purification, Staining, Affinity Purification, SDS Page, Plasmid Preparation, Transfection, Negative Control, Over Expression, Control

Journal: BMC biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: Figure 4 The immunohistochemistry staining on NSCLC tissue sections with rabbit monoclonal anti-PCYT1A antibody or 8F1 mouse monoclonal anti-ERCC1 antibody. Pathologist-validated NSCLC FFPE tissue blocks were cut into thin sections, and then further deparaffinized, re-hydrated before IHC testing. The primary antibodies (Left: rabbit anti-PCYT1A mAb, Right: 8F1 anti-ERCC1 mAb) used for these experiments were diluted at 1:150 dilution with blocking buffer.

Article Snippet: Primers used for ERCC1 mRNA detection were HP229725 with 50- GCTGGCTAAGATGTGTATCCTGG-30 (forward) and 5-ATCAGGAGGTCCGCTGGTTTCT-3 (reverse), for PCYT1A were HP208235 with 50-GTTCCTTCCAAA GTGCAGCGCT-30 (forward) and 5-AGGAGTTCCTC TGCTGGCTTCT-3, for β-actin were HP204660 with 50-CAGCCATGTACGTTGCTATCCAGG-30 (forward) and 5-AGGTCCAGACGCAGGATGGCATG-3 (OriGene Technologies Inc.).

Techniques: Immunohistochemistry, Staining, Blocking Assay

Journal: BMC biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: Figure 6 To develop the most highly specific anti-ERCC1 monoclonal antibody with a protein microarray chip. A. Specificity evaluation with a protein microarray chip. The overexpression protein microarray chip was immunostained with the 4F9 clone. This data shows that 4F9 is highly specific to ERCC1 (indicated with red arrows). No cross-reactivity was observed with any other test protein. B. Western Blot confirmation analysis. Seven OriGene VERIFYTM overexpression lysate antigen standards (Lane 1 to 7) were fractionated on SDS-PAGE, and then immunoblotted with 4F9. The recombinant protein expression levels for the each of the different protein overexpression lysates were confirmed with the anti-DDK antibody (Figure 2B). Lanes 1 to 7 are loaded with samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and PCYT1A (NM_005017).

Article Snippet: Primers used for ERCC1 mRNA detection were HP229725 with 50- GCTGGCTAAGATGTGTATCCTGG-30 (forward) and 5-ATCAGGAGGTCCGCTGGTTTCT-3 (reverse), for PCYT1A were HP208235 with 50-GTTCCTTCCAAA GTGCAGCGCT-30 (forward) and 5-AGGAGTTCCTC TGCTGGCTTCT-3, for β-actin were HP204660 with 50-CAGCCATGTACGTTGCTATCCAGG-30 (forward) and 5-AGGTCCAGACGCAGGATGGCATG-3 (OriGene Technologies Inc.).

Techniques: Microarray, Over Expression, Western Blot, SDS Page, Recombinant, Expressing

Journal: Scientific Reports

Article Title: Stimulated phospholipid synthesis is key for hepatitis B virus replications

doi: 10.1038/s41598-019-49367-8

Figure Lengend Snippet: The relative mRNA expression levels of major genes in PC synthesis and metabolic pathways. ( A ) Phosphatidylcholine synthesis and metabolism pathways. ( B ) The relative mRNA level of major genes in phosphatidylcholine synthesis and metabolism in HepG2 and HepG2.2.15 cells, n = 3, detailed data are shown in Table . ( C ) The western blot analysis of LPP1, PCYT1A protein expression levels in HepG2 and HepG2.2.15 cells, the initial figure is shown in Fig. . ( D ) The levels of the substrates of the PC synthesis in HepG2 and HepG2.2.15 cells, n = 10. ( B , D ) data are shown as mean ± SD, t-test, *** p value < 0.001, ** p value < 0.01, * p value < 0.05. PA: phos p hatidic acid, DAG: diglyceride, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PS: phosphatidylserine, LPC: lyso-phosphatidylcholine, GPC: glycerol-phosphorylcholine.

Article Snippet: The PCYT1A siRNA, LPP1 siRNA and negative control siRNA used in the experiment were obtained from GenePharma (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Stimulated phospholipid synthesis is key for hepatitis B virus replications

doi: 10.1038/s41598-019-49367-8

Figure Lengend Snippet: HepG2.2.15 cells were transfected by siRNA. ( A ) The framework of gene silencing with siRNA. ( B ) The relative mRNA levels of PCYT1A and LPP1 . ( C ) The western blot analysis of LPP1, PCYT1A protein expression levels in the siRNA negative control group and treatment group, the initial figure is shown in Fig. . ( D ) The relative levels of HBsAg. ( E ) The relative levels of HBeAg. ( F ) The relative levels of HBV DNA. ( G ) The proliferation of HepG2.2.15 cells transfected by siRNA was assayed using cell-counting assay. ( B , D – F ) data are shown as mean ± SD, n = 3, t-test *** p value < 0.001, ** p value < 0.01, * p value < 0.05. PA: phos p hatidic acid, DAG: diglyceride, PC: phosphatidylcholine. siRNA: si PCYT1A or si LPP1 , siNC: negative control siRNA.

Article Snippet: The PCYT1A siRNA, LPP1 siRNA and negative control siRNA used in the experiment were obtained from GenePharma (Shanghai, China).

Techniques: Transfection, Western Blot, Expressing, Negative Control, Cell Counting

Journal: Communications Biology

Article Title: Glutathione S-transferase Mu 2 inhibits hepatic steatosis via ASK1 suppression

doi: 10.1038/s42003-022-03251-w

Figure Lengend Snippet: a – c PCYT1A expression level was down-regulated by GSTM2 KD and up-regulated by GSTM2 OE; * p < 0.05, ** p < 0.01. d PCYT1A KD induced LD expansion. e , f GSTM2 affected recruitment of GPAT4 and DGAT2 on LD surface (in the presence of oleic acid medium), bar, 10 μm, 1 μm (inlay). g Expression level of GPAT4 and DGAT2 was regulated by treatment of palmitic acid (ASK1 activator) and GS-4997 (ASK1 activity inhibitor). h Recruitment of GPAT4 on LD surface was regulated by palmitic acid or GS-4997 treatment, bar, 10 μm, 1 μm (inlay). i , j qPCR detection of PCYTA1 expression with PA or GS-4997 treatment respectively.

Article Snippet: The following rabbit polyclonal antibodies were used: anti-GSTM2 (#ab125,102, Abcam, Cambridge, UK, 1:1000), anti-GSTM2 (#A13,496, ABclonal, Wuhan, China, 1:2000), anti-GAPDH (#CSB-PA00025A0Rb Flarebio Biotech LLC, College Park, MD, USA, 1:1000), anti-CIDEC (#D222114, Sangon Biotech, Shanghai, China, 1:1000), anti-CIDEC (#12287-1-AP, Proteintech, Rocky Hill, NJ, USA, 1:1000), anti-ADRP/perilipin2 (#15294-1-AP, Proteintech), anti-DGAT2 (#bs-12,998R, BIOSS, Beijing, China), anti-DGAT2 (#A13,891, ABclonal, Wuhan, China, 1:2000), anti-GPAT4 (#bs-15,587R, BIOSS), anti-SREBP1 (#14,088-1-AP, Proteintech), and anti-PCYT1A (#bs-11306R, BIOSS), anti-PCYT1A (#A5935, ABclonal, Wuhan, China, 1:1000).

Techniques: Expressing, Activity Assay

Journal: Communications Biology

Article Title: Glutathione S-transferase Mu 2 inhibits hepatic steatosis via ASK1 suppression

doi: 10.1038/s42003-022-03251-w

Figure Lengend Snippet: GSTM2 was highly up-regulated in hepatic steatosis tissues. As the member of phase II drug metabolizing enzymes, GSTM2 could detoxify the toxicity of metabolic products such as ROS. ROS could enhance the inflammation and fibrosis progression. Furthermore, GSTM2 interacted with ASK1 directly and suppressed ASK1 phosphorylation. Therefore, GSTM2 down-regulated expression of SREBP and perilipins to inhibit the LD accumulation. ASK1 signaling also regulated the expression level of DGAT2, GPAT4 and PCYT1A and the recruitment of DGAT2 and GPAT4 on LD surface to modulate the LD growth. In conclusion, GSTM2 protects against the hepatic steatosis of NAFLD via its antioxidant and suppressing ASK1-p38/JNK signaling.

Article Snippet: The following rabbit polyclonal antibodies were used: anti-GSTM2 (#ab125,102, Abcam, Cambridge, UK, 1:1000), anti-GSTM2 (#A13,496, ABclonal, Wuhan, China, 1:2000), anti-GAPDH (#CSB-PA00025A0Rb Flarebio Biotech LLC, College Park, MD, USA, 1:1000), anti-CIDEC (#D222114, Sangon Biotech, Shanghai, China, 1:1000), anti-CIDEC (#12287-1-AP, Proteintech, Rocky Hill, NJ, USA, 1:1000), anti-ADRP/perilipin2 (#15294-1-AP, Proteintech), anti-DGAT2 (#bs-12,998R, BIOSS, Beijing, China), anti-DGAT2 (#A13,891, ABclonal, Wuhan, China, 1:2000), anti-GPAT4 (#bs-15,587R, BIOSS), anti-SREBP1 (#14,088-1-AP, Proteintech), and anti-PCYT1A (#bs-11306R, BIOSS), anti-PCYT1A (#A5935, ABclonal, Wuhan, China, 1:1000).

Techniques: Expressing