Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 1. Overexpression of mutant α-synuclein (α-syn) E46K attenuated neurite outgrowth. We transfected SK-N-SH cells with human wild-type (WT) α-syn, A53T or E46K mutant α-syn, or an empty

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Over Expression, Mutagenesis, Transfection

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 2. Proliferation of mutant α-syn E46K transfectants. We performed cell proliferation assays using the alamarBlue reagent for 72 h after transfection with pcDNA3.1+-α-syn (WT, A53T, or E46K). The fluorescence of the alamarBlue indicator (excitation, 530 nm; emission, 590 nm) was analyzed using a microplate fluorometer. All samples were prepared in triplicate. Data are presented as means ± SD.

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Mutagenesis, Transfection, Fluorescence

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 3. Cell division control 42 effector protein 2 (Cdc42EP2) negatively regulated neurite outgrowth by downregulating βIII-tubulin in E46K α-syn transfectants. (A) Quantitative analysis of Cdc42EP2 mRNA expression. SK-N-SH cells were seeded into six-well plates (1 × 105 cells/well) and cultured for 16 h to achieve attachment and confluency. After transfection with each pcDNA3.1+-α-syn (WT, A53T, and E46K), the cells were maintained at 37 ◦C in an atmosphere of 5% CO2 for 24 h. Then, total RNA was isolated for cDNA synthesis, followed by real-time quantitative PCR using SYBR Green TOPrealTM qPCR PreMIX (Enzynomics) on an Eco Real-Time PCR System (Illumina). Data are presented as means ± SD from four independent experiments. (B) Quantitative analysis of βIII- tubulin gene expression using the same techniques and statistical analyses as described in Figure 3A. (C) Western blot analysis of Cdc42EP2 protein levels in α-syn transfectants, including the empty vector group (control). (D) Western blot analysis of βIII-tubulin levels in α-syn transfectants, including the empty vector group (control). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of prepared cell lysate samples and transferring the proteins onto membranes, western blotting was performed using specific antibodies for human α-syn, Cdc42EP2, or βIII-tubulin. Figure S1 presents representative blots from three independent experiments. Data are presented as means ± SD for three independent experiments performed in triplicate. Analysis was performed using one-way ANOVA (* p < 0.05, ** p < 0.01) and Student t-test (# p < 0.05, ## p < 0.01).

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Control, Expressing, Cell Culture, Transfection, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Gene Expression, Western Blot, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Transferring

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 4. α-Syn-induced Cdc42EP2 expression regulated neurite outgrowth in SK-N-SH cells. Confocal microscopy analysis of SK-N-SH cells transfected with WT, A53T, and E46K α-syn, including empty

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Expressing, Confocal Microscopy, Transfection

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 5. Cdc42EP2 knockdown abrogated α-syn-induced neurite outgrowth. Small interfering RNA (siRNA) targeting Cdc42EP2 was transfected into SK-N-SH cells. Unrelated siRNA was used as a

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Knockdown, Small Interfering RNA, Transfection

Journal: Journal for immunotherapy of cancer

Article Title: C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.

doi: 10.1136/jitc-2025-011472

Figure Lengend Snippet: Figure 1 Complement C5a/C5aR pathway was activated in breast cancer tissue and associated with malignant degrees of breast cancer cells. (A) UMAP plot showing the overview of 9 cell clusters of 83,052 cells derived from normal human breast and breast cancer tissues across four datasets. (B) Dot plot showing the representative marker genes across the nine clusters. (C) Gene expression visualized by UMAP plot for marker genes of epithelial cells. (D) GO analysis of differently expressed genes in cells from the breast cancer group versus the normal group. (E) Violin plot showing distribution of C5a/C5aR pathway- related gene expression of normal and tumor tissues. Asterisks indicate statistical significance among groups using two-sided Wilcoxon test. (F) Immunohistochemistry and quantitative analysis showing the expression distribution of C5aR in normal and breast cancer tissue from patients. Statistical differences are assessed by unpaired t-test. (G) Heatmaps showing the expression distribution of C5aR1-related key transcription factors involved in differential transcription activity between normal and breast cancer tissue. (H) UMAP plot showing 18 clusters of epithelial cells. (I) Violin plots show the complement activation score in 18 epithelial cell clusters. The red line displays the selected complement activation score threshold; the third quartile was selected for comparison with the threshold. (J) Violin plots show the CNV level in 18 epithelial cell clusters. (K) UMAP plot showing high and low complement activation clusters of epithelial cells. (L) Violin spots of CNV level in the high and low complement activation group. Data were mean±SEM. ***p<0.001; ****p<0.0001. CNV, copy number variation; C5aRA, C5a receptor antagonists; GO, Gene Ontology; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1- AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1- AP), LIAS (Proteintech, 11577–1- AP), β-catenin (Proteintech, 51067–2- AP), ATP7B (Proteintech, 19786–1- AP), C5b- 9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1- AP), HRP- labeled Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP- labeled Goat Anti- Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0423).

Techniques: Derivative Assay, Marker, Gene Expression, Immunohistochemistry, Expressing, Activity Assay, Activation Assay, Comparison

Journal: Journal for immunotherapy of cancer

Article Title: C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.

doi: 10.1136/jitc-2025-011472

Figure Lengend Snippet: Figure 2 C5a/C5aR pathway was related to breast cancer cell cuproptosis. (A) GO analysis of differently expressed genes in epithelial cells from the breast cancer group versus the normal group. (B) Violin plots show the cuproptosis score in 18 epithelial cell clusters. (C) Association between complement activation score (cited from BP entry in GO enrichment) and cuproptosis score analyzed by Spearman’s analysis. (D) ssGSEA analysis of cuproptosis in epithelial cells derived from tumor and normal tissue. (E) Cuproptosis score, complement activation score, and cells with both high scores visualized by UMAP plot of epithelial cells. (F) Potential trajectory of all epithelial cells identified two distinct cell fates colored by cluster. (G) Heatmap showing selected-related genes of cuproptosis and C5-C5aR pathway along the pseudotime, which was clustered into three profiles. Color keys differentially coding from blue to red indicated the relative expression levels from low to high. (H) Dot plots of dynamic expression of key genes in cuproptosis and C5-C5aR pathway along two cell fates. (I) PPI network shows potentially existing connections between complement-related genes, copper transporters genes and cuproptosis-related genes. (J) GSEA analysis of various forms of programmed cell death (PCD) in 4T1 cells treated with C5a. (K–L) Volcano plot and heatmaps of differentially expressed genes between control groups and C5a treated groups in 4T-1 cells. (M–N) Western blotting and RT-qPCR analysis of ATP7B expression in 4T1 cells between control and C5a treated groups. (O) GSEA analysis of various forms of PCD in MDA-MB-231 cells treated with C5a. (P–Q) Volcano plot and heatmaps of differentially expressed genes between control groups and C5a treated groups in MDA-MB-231 cells. (R–S) Western blotting and RT-qPCR analysis of ATP7B expression in MDA-MB-231 cells between control and C5a-treated groups. Statistical differences are assessed by unpaired t- test. Data were mean±SEM. *p<0.05; **p<0.01. BP, Biological Process; C5aRA, C5a receptor antagonists; FDR, False Discovery Rate; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; RT-qPCR, real-time quantitative PCR; ssGSEA, Single-Sample Gene Set Enrichment Analysis; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1- AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1- AP), LIAS (Proteintech, 11577–1- AP), β-catenin (Proteintech, 51067–2- AP), ATP7B (Proteintech, 19786–1- AP), C5b- 9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1- AP), HRP- labeled Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP- labeled Goat Anti- Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0423).

Techniques: Activation Assay, Derivative Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.

doi: 10.1136/jitc-2025-011472

Figure Lengend Snippet: Figure 3 C5a/C5aR pathway mediated cuproptosis resistance by upregulating the expression of ATP7B via activation of the Wnt/β-catenin pathway. (A) Morphological changes in 4T1 cells after different treatments (Ctrl, ES-Cu, ES-Cu+C5 a, ES- Cu+C5 a +C5 aRA). Scale bar: 100 µm. (B) Cell viability of 4T1 cells with indicated drugs. (C) Representative microscopy images of DLAT aggregation in 4T1 cells after the indicated treatment. The white arrows indicate DLAT aggregation. Scale bars: 40 µm. (D) Western blot of 4T1 cells treated with indicated drugs. (E) Heatmap showing the expression distribution of key genes of the Wnt/β-catenin pathway in different groups of 4T1 cells. (F) Heatmap showing the expression distribution of key genes of Wnt/β-catenin pathway in different groups of MDA-MB-231 cells. (G) Real-time quantitative PCR of 4T1, E0771, MDA-MB-231 and MCF7 cells treated with indicated drugs. (H) Western blot of 4T1, E0771, MDA-MB-231 and MCF7 cells treated with indicated drugs. Statistical differences are assessed by unpaired t-test. Data were mean±SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Ca5RA, C5a receptor antagonists; DLAT, dihydrolipoamide S-acetyltransferase; ES-Cu, elesclomol-Cu; mRNA, messenger RNA.

Article Snippet: The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1- AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1- AP), LIAS (Proteintech, 11577–1- AP), β-catenin (Proteintech, 51067–2- AP), ATP7B (Proteintech, 19786–1- AP), C5b- 9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1- AP), HRP- labeled Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP- labeled Goat Anti- Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0423).

Techniques: Expressing, Activation Assay, Microscopy, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.

doi: 10.1136/jitc-2025-011472

Figure Lengend Snippet: Figure 5 In vivo toxicity evaluation of CuS NPs and C5a/C5aR pathway blocking enhanced the antitumor effect of CuS NPs on 4T1 cells. (A) Levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREA (serum creatinine), TBIL (total bilirubin), DBIL (direct bilirubin), UREA (urine creatinine) in serum. (B) H&E-stained images of major organs after indicated treatments. Scale bars: 100 µm. (C) ELISA assay of C5a in control and CuS NPs treated groups. (D) Immunohistochemistry revealed the expression of C5b-9 and C5aR in tumor tissues from 4T1 xenografts (left) and 4T1 xenografts (right) with CuS NPs and quantitative analysis. Scale bars: 50 µm. (n=3/group, Statistical differences are assessed by unpaired t-test. Data were mean±SEM. *p<0.05; **p<0.01; ***p<0.001). (E) Representative morphological changes in 4T1 cells after different treatments. Scale bar: 100 µm. (F–G) Colony formation assay of 4T1 cells treated with indicated drugs and quantitative analysis. (H) Cell viability of 4T1 cells with indicated drugs. (I) Representative confocal microscopy images of DLAT aggregation in 4T1 cells after the indicated treatment. The white arrows indicate DLAT aggregation. Scale bars: 40 µm. (J) Relative content analysis of ROS in 4T1 cells stained with DCFH-DA fluorescent probes after various treatments. (K) Western blot result of LIAS in 4T1 cells after the indicated treatment for 48 hours. Statistical differences are assessed by unpaired t-test. Data were mean±SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. C5aRA, C5a receptor antagonists; DAPI, 4',6-Diamidino-2-phenylindole; DLAT, dihydrolipoamide S-acetyltransferase; IHC, immunohistochemistry; LIAS, lipoic acid synthase; NPs, nanoparticles; ROS, reactive oxygen species.

Article Snippet: The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1- AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1- AP), LIAS (Proteintech, 11577–1- AP), β-catenin (Proteintech, 51067–2- AP), ATP7B (Proteintech, 19786–1- AP), C5b- 9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1- AP), HRP- labeled Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP- labeled Goat Anti- Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0423).

Techniques: In Vivo, Blocking Assay, Staining, Enzyme-linked Immunosorbent Assay, Control, Immunohistochemistry, Expressing, Colony Assay, Confocal Microscopy, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.

doi: 10.1136/jitc-2025-011472

Figure Lengend Snippet: Figure 6 Blocking the C5a/C5aR pathway enhanced the antitumor efficacy of CuS NPs in the 4T1 xenograft mouse model. (A) Balb/c mice were implanted with 3.0×105 4T1 cancer cells in the mammary fat pad with and without CuS NPs/lazer and C5aRA on the 5th day and 8th day. The tumor growth was measured by representative tumor images (B), tumor size (C), and tumor weight (D). (E) Immunohistochemistry revealed the expression of LIAS in tumor tissues from 4T1 xenografts mouse with indicated treatment. (F) Representative flow cytometry plots and the corresponding percentages of the CD11b+ LY-6G+ MDSCs population in tumor tissues from mice subjected to various treatments (n=3). (G) Representative flow cytometry plots and the corresponding percentages of the CD11C+ /IA-IE DC cells population in tumor tissues from mice subjected to various treatments (n=3). (H) The assessment of main organs by H&E staining. Statistical differences are assessed by one-way analysis of variance. Data were mean±SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. C5aRA, C5a receptor antagonists; DC, dendritic cell; IHC, immunohistochemistry; LIAS, lipoic acid synthase; MDSCs, myeloid-derived suppressor cells; NPs, nanoparticles.

Article Snippet: The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1- AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1- AP), LIAS (Proteintech, 11577–1- AP), β-catenin (Proteintech, 51067–2- AP), ATP7B (Proteintech, 19786–1- AP), C5b- 9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1- AP), HRP- labeled Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP- labeled Goat Anti- Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0423).

Techniques: Blocking Assay, Immunohistochemistry, Expressing, Flow Cytometry, Staining, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.

doi: 10.1136/jitc-2025-011472

Figure Lengend Snippet: Figure 7 Blocking the C5a/C5aR pathway enhanced the antitumor efficacy of CuS NPs in multiple tumor models. (A) Mice were implanted with 3.0×105 B16-F10 cancer cells under the skin with and without CuS NPs/lazer and C5aRA on the 5th day and 8th day. The tumor growth was measured by representative tumor images (B), tumor size (C) and tumor weight (D). (E) Mice were implanted with 3.0×105 CT-26 cancer cells under the skin with and without CuS NPs/lazer and C5aRA on the 5th day and 8th day. The tumor growth was measured by representative tumor images (F), tumor size (G), and tumor weight (H). (I) Schematic of mechanisms that C5a/C5aR pathway blocking enhanced antitumor effect of CuS nanoparticles. Statistical differences are assessed by one-way analysis of variance. Data were mean±SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. C5aRA, C5a receptor antagonists; DC, dendritic cell; DLAT, dihydrolipoamide S-acetyltransferase; Fe-S, iron-sulfur; MDSCs, myeloid-derived suppressor cells; NPs, nanoparticles; ROS, reactive oxygen species; TCA, tricarboxylic acid; TME, tumor microenvironment.

Article Snippet: The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1- AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1- AP), LIAS (Proteintech, 11577–1- AP), β-catenin (Proteintech, 51067–2- AP), ATP7B (Proteintech, 19786–1- AP), C5b- 9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1- AP), HRP- labeled Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP- labeled Goat Anti- Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti- Rabbit IgG (H+L) (Beyotime, Cat#A0423).

Techniques: Blocking Assay, Derivative Assay