Journal: Theranostics

Article Title: Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes

doi: 10.7150/thno.80801

Figure Lengend Snippet: Antibodies used for immunostaining

Article Snippet: Rabbit anti-myosin light chain ventricular isoform , 1:600 , 10906-1-AP , Proteintech , Goat anti-rabbit IgG-FITC , 1:800 , A32790 , Thermo Fisher.

Techniques:

Journal: Theranostics

Article Title: Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes

doi: 10.7150/thno.80801

Figure Lengend Snippet: Antibodies used for fluorescence activated cell sorting

Article Snippet: Rabbit anti-myosin light chain ventricular isoform , 1:600 , 10906-1-AP , Proteintech , Goat anti-rabbit IgG-FITC , 1:800 , A32790 , Thermo Fisher.

Techniques: Fluorescence

Journal: Theranostics

Article Title: Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes

doi: 10.7150/thno.80801

Figure Lengend Snippet: Antibodies used in Western Blot analysis

Article Snippet: Rabbit anti-myosin light chain ventricular isoform , 1:600 , 10906-1-AP , Proteintech , Goat anti-rabbit IgG-FITC , 1:800 , A32790 , Thermo Fisher.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A DLD1 cells were treated with MG132 (20 μM) for 5 h, lysed and subjected to immunoprecipitation with anti-PRPF19 antibody or IgG using silver staining. The red arrow represents the detected bands. The potential interacting proteins of PRPF19 that possessed the highest score were listed (right panel). B The DLD1 cells were co-transfected with the indicated plasmids for 48 h, and cells were treated with MG132 for 5 h. The cell lysates were immunoprecipitated with antibody anti-Flag and immunoblotted with anti-HA (left panel); immunoprecipitated with antibody anti-HA and immunoblotted with anti-Flag (right panel). C DLD1 cells were treated with MG132 for 5 h before being collected. Cell lysates were immunoprecipitated with anti-IgG, anti-MYL9, or anti-PRPF19 followed by immunoblot. D , E The mRNA ( D ) and protein ( E ) expression level of MYL9 in the DLD1 stable cell lines. F , G The indicated stable cell lines with PRPF19 knockdown (scramble/shPRPF19) or overexpression (Ctrl/PRPF19) were treated with CHX, and lysed for WB analysis at the specific time points. The intensity of MYL9 expression for each time point was quantified with GAPDH as a normalizer. *** p < 0.001, ** p < 0.01 based on Student’s t test.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Immunoprecipitation, Silver Staining, Transfection, Western Blot, Expressing, Stable Transfection, Knockdown, Over Expression

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A , B The indicated stable cell lines with PRPF19 overexpression (Ctrl/PRPF19) or knockdown (scramble/shPRPF19) were treated with MG132 or DMSO as control. Cell lysates were analyzed by immunoblot. C HEK293T cells were transfected with siRNA of PRPF19 for 24 h, and then co-transfected with HA-MYL9 and His-Ub plasmids for another 24 h. After treatment with MG132 for 5 h, cell lysates were collected for co-IP and subjected to immunoblot analysis. D In vivo poly-ubiquitination of MYL9. HEK293T cells were co-transfected with plasmids of MYL9, PRPF19 and WT Ub or Ub mutants (K48O, K63O) for 48 h, followed by MG132 treatment for 5 h before lysis. Immunoprecipitation of ubiquitin-conjugated MYL9 protein was performed with anti-His antibody. Immuno-complexes and input were analyzed by immunoblot. E HEK293T cells were transfected with the plasmids of MYL9, PRPF19 and WT Ub or Ub mutants (K48R, K63R) for 48 h. Cells were treated with MG132 and lysed for immunoprecipitation and immunoblot as described in ( D ). F The in vitro ubiquitination assays were performed and the western blot assays with the indicated antibody. G , H Representative IHC staining images for PRPF19 and MYL9 of different staining intensities ( G ), and the correlation between the expression of PRPF19 and MYL9 based on the H-score in CRC patient tissues ( n = 30) were analyzed ( H ).

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Stable Transfection, Over Expression, Knockdown, Control, Western Blot, Transfection, Co-Immunoprecipitation Assay, In Vivo, Ubiquitin Proteomics, Lysis, Immunoprecipitation, In Vitro, Immunohistochemistry, Staining, Expressing

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A – D MYL9 was transiently suppressed by transfecting siRNA into cells, or overexpressed by transfecting plasmids pReceiver-MYL9 (pR-MYL9) or pReceiver (pR) into the indicated cell lines. Representative images of transwell ( A , B ) and wound healing ( C , D ) assays were shown (upper panel), and quantification was presented (lower panel). The means ± SD of triplicate samples were shown. E The livers were collected, and representative images of liver metastatic nodules of HE-stained sections highlighted by arrows were shown. F The number of liver metastatic nodules was analyzed. ** p < 0.01, * p < 0.05 based on Student’s t test.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A , B The mRNA expression of YAP1 and its downstream targets were detected by qRT-PCR. C , D The DLD1 cells stably overexpressed PRPF19 were transfected with siRNA of MYL9, and HCT116 cells stably interfered with PRPF19 were transfected with MYL9 plasmids pReceiver-MYL9 (pR-MYL9) or pReceiver (pR). The indicated molecules were analyzed by WB. *** p < 0.001 based on Student’s t test.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, Transfection

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: PRPF19 enhanced the stability of MYL9 via K63-linked ubiquitination, and the Src-YAP1 pathway acts as the downstream effector mechanism of the PRPF19/MYL9 axis in CRC metastasis.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics

Journal: Journal of dairy science

Article Title: Yak milk promotes renal calcium reabsorption in mice with osteoporosis via the regulation of TRPV5.

doi: 10.3168/jds.2022-23218

Figure Lengend Snippet: Figure 4. Effect of yak milk on immunohistochemistry of kidney in mice. (A) Images of mouse kidney tissue cross sections immunostained for TRPV5 and calbindin-D28k (scale bar = 100 μm). (B) Relative mean density of TRPV5 in mouse kidney. (C) Relative mean density of calbindin-D28k in mouse kidney. Mean density = (integrated optical density [IOD] sum)/area. L-YM = low-dose yak milk group, 1,000 mg/kg per day; H-YM = high-dose yak milk group, 2,000 mg/kg per day; ALN = alendronate sodium group, 1 mg/kg per day. Data are expressed as mean ± SD. Means with different lowercase letters (a–d) in a column are significantly different (P < 0.05).

Article Snippet: The anti-TRPV5 antibody (M03218), anticalbindinD28k antibody (PB9045) and hypersensitive chemiluminescence kit (AR1174) were procured from BOSTER Biological Technology Co. Ltd.

Techniques: Immunohistochemistry

Journal: Journal of dairy science

Article Title: Yak milk promotes renal calcium reabsorption in mice with osteoporosis via the regulation of TRPV5.

doi: 10.3168/jds.2022-23218

Figure Lengend Snippet: Figure 5. Effects of yak milk on the expression of calcium transporters TRPV5 and calbindin-D28k in mouse kidney. (A) Western blot showing the expression of TRPV5 and calbindin-D28k in mouse kidney. (B) Quantification of TRPV5 expression from the Western blots. (C) Quantification of calbindin-D28k expression from the Western blots. L-YM = low-dose yak milk group, 1,000 mg/kg per day; H-YM = high-dose yak milk group, 2,000 mg/kg per day; ALN = alendronate sodium group, 1 mg/kg per day. Data are expressed as mean ± SD. Means with dif- ferent lowercase letters (a–c) in a column are significantly different (P < 0.05).

Article Snippet: The anti-TRPV5 antibody (M03218), anticalbindinD28k antibody (PB9045) and hypersensitive chemiluminescence kit (AR1174) were procured from BOSTER Biological Technology Co. Ltd.

Techniques: Expressing, Western Blot

Journal: Journal of dairy science

Article Title: Yak milk promotes renal calcium reabsorption in mice with osteoporosis via the regulation of TRPV5.

doi: 10.3168/jds.2022-23218

Figure Lengend Snippet: Figure 6. Effects of yak milk on the expression of calcium transporters TRPV5 and calbindin-D28k mRNA in mouse kidney. (A) Relative expression of TRPV5 mRNA in mouse kidney. (B) Relative expression of calbindin-D28k mRNA in mouse kidney. L-YM = low-dose yak milk group, 1,000 mg/kg per day; H-YM = high-dose yak milk group, 2,000 mg/kg per day; ALN = alendronate sodium group, 1 mg/kg per day. Data are expressed as mean ± SD. Means with different lowercase letters (a–d) in a column are significantly different (P < 0.05).

Article Snippet: The anti-TRPV5 antibody (M03218), anticalbindinD28k antibody (PB9045) and hypersensitive chemiluminescence kit (AR1174) were procured from BOSTER Biological Technology Co. Ltd.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Identification of MYLK3 mutations in familial dilated cardiomyopathy

doi: 10.1038/s41598-017-17769-1

Figure Lengend Snippet: Western blot analysis suggested decreased expression levels of mutant cMLCK and MLC2 phosphorylation. ( a ) Expression of wild-type and mutant cMLCK protein in transfected HEK293T cells. Western blot analysis showed the reduced expression of mutant cMLCK protein. The blots were run under the same experimental conditions. The uncropped images are in Supplementary Fig. . ( b ) Densitometric quantification of western blots. Data are expressed as mean ± SEM (N = 3). * P < 0.05. ( c ) Wild-type or mutant MYLK3 transfected HEK293T cells were treated with cycloheximide (CHX) for indicated times. Western blot analysis showed rapid reduction of mutant proteins. Data are expressed as mean ± SEM (N = 3). * P < 0.05. The blots were run under the same experimental conditions. The uncropped images are in Supplementary Fig. . ( d ) Phos-tag SDS-PAGE followed by western blot analysis showed phosphorylated and non-phosphorylated forms of MLC2 using anti-FLAG antibody. Levels of phosphorylated MLC2 were remarkably reduced when co-transfected with mutant MYLK3 and MYL2 constructs. The ratio of phosphorylated MLC2 to total MLC2 (phosphorylated MLC2+ non-phosphorylated MLC2) was determined. Data are expressed as mean ± SEM (N = 3). * P < 0.05. The uncropped images are in Supplementary Fig. .

Article Snippet: A full-length cDNA clone of human MYL2 (NM_000432) with an C-terminal Myc-FLAG-Tag was obtained from Origene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Mutagenesis, Transfection, SDS Page, Construct