Journal: Cell Genomics

Article Title: Continuous transcriptome analysis reveals novel patterns of early gene expression in Drosophila embryos

doi: 10.1016/j.xgen.2023.100265

Figure Lengend Snippet:

Article Snippet: The resulting aRNA was fragmented for exactly 3 min at 94°C using NEBNext Magnesium RNA Fragmentation Module (5 μL fragmentation buffer plus 5 μL stop solution per reaction, NEB, USA). aRNA was cleaned up with 0.8 μL RNAClean XP (Beckman Coulter, USA) per 1 μL sample, recovering a total of 21 μL aRNA.

Techniques: Recombinant, Reverse Transcription, Software

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: Downregulation of BAG3 affects p-STAT3(705) and p-STAT3(727)

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 regulates p-STAT3(705) and p-STAT3(727) through p-JAK2 and p-ERK1/2 respectively to promote VSMCs migration

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques: Migration

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 modulates STAT3 phosphorylation by regulating the interaction of STAT3 with JAK2 and ERK1/2

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Neoplasia (New York, N.Y.)

Article Title: Synergism of BARF1 with Ras Induces Malignant Transformation in Primary Primate Epithelial Cells and Human Nasopharyngeal Epithelial Cells 1

doi:

Figure Lengend Snippet: (A) Detection of H-Ras, c-myc, and Bcl-2 by Western blot analysis in primary PATAS cells, BARF1-transfected PATAS (P-BA), BARF1 and Ras-transfected PATAS (P-BA-R), hTERT-transfected PATAS (P-TE), and hTERT and Ras-transfected PATAS cells (P-TE-R). (B) Foci formation was detected by light microscopy. Whereas BARF1-transfected PATAS cells showed an organized growth (photographed at x50 [a] and x100 [c]), PATAS expressing both BARF1 and Ras genes formed a higher number of large foci (photographed at x50 [b] and x100 [d]). Cells were cultured in DMEM supplemented with 10% serum. (C) Schematic diagram of the SV40 ER and the alternatively spliced SV40 viral transcripts coding for large T, small T, and 17K T proteins. Numbering refers to the SV40 genomic nucleotide numbers. Origin of replication was indicated as Ori. (D) PCR for the detection of the alternatively spliced messenger RNA encoding the SV40 ST. Positions of PCR primers are indicated in panel A. m, marker. a, PCR with NP69 cell DNA to amplify SV40 ER including LT exons and intron. b, RT-PCR with NP69 cell RNA to amplify SV40 LT transcript. c, RT-PCR with NP69 cell RNA to amplify SV40 ST transcript. d, RT-PCR with 293T cell RNA to amplify SV40 LT transcript. e, PCR with 293 cell DNA as negative control.

Article Snippet: Western Blot Analysis Immunoblot analysis was performed as previously described [ 18 ]; after separation by 8% to 12% polyacrylamide gel electrophoresis, the following antibodies were used to confirm protein expression: primary antibody against Bcl-2 (sc-7382; Santa Cruz, Germany), c- myc (sc-764; Santa Cruz), BARF1 (anti-Pep 2A serum, a polyclonal rabbit antiserum prepared against a synthetic peptide corresponding to a presumed epitope, amino acids (aa) 172 to 180 [NGGVMKEKD], of the BARF1 protein), Ras (sc-29; Santa Cruz), SV40 Tantigen (sc-148; Santa Cruz; N-terminal epitope mapping within residues 1 to 82 of SV40 large Tantigen for detection of 94-kDa SV40 LT antigen and 21-kDa SV40 ST antigen), LMP1 (BD Biosciences Pharmingen, San Diego, CA), h-TERT (SC-7212; Santa-Cruz), and tubulin (sc-5286; Santa Cruz).

Techniques: Western Blot, Transfection, Light Microscopy, Expressing, Cell Culture, Marker, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Synergism of BARF1 with Ras Induces Malignant Transformation in Primary Primate Epithelial Cells and Human Nasopharyngeal Epithelial Cells 1

doi:

Figure Lengend Snippet: (A) Expression of BARF1 protein and H-Ras in cell lines, tumor biopsies, and tumor cell lines. Cell extracts were prepared from PATAS (primary cells), P-BA (PATAS transfected by BARF1), P-BA-R (P-BA plus Ras), P-BA-R-T (tumor biopsy induced by injection of P-BA-R), and P-BA-R-TL (established cell line from tumor biopsy induced by injection of P-BA-R). For BARF1 detection, A20, BARF1 protein produced by BARF1 recombinant adenovirus system was used as a positive control. For H-Ras detection, we used protein extracted from NPC biopsy expressing a high Ras protein (NPC26) as a positive control. (B) Detection of cytokeratin AE1/AE3 in cell lines. Cells were cultured in microchamber for 72 hours and fixed with acetone for 15 minutes, then anti-AE1/AE3 with a dilution of 50 was treated on each cell. a, Primary PATAS cells. b, P-BA-R cell line. c, P-BA-R tumor cell culture after 4 days (TL1). d, The 30th passage of P-BA-R tumor cell line (TL1). (C) Western blot analysis of associated signaling molecules in BARF1-expressing, LMP1-expressing, and control cell populations. Different cell populations were indicated at the top of the panel. The detected proteins are indicated on the right side. (D) RT-PCR detection of BARF1 and Western blot analysis of Ras and SV40 LT and ST expressions in cloned cell line and tumors. Different tumors formed by NP69-BARF1 + Ras cloned cell line were indicated at the top of the panel. The detected molecules are indicated on the right side.

Article Snippet: Western Blot Analysis Immunoblot analysis was performed as previously described [ 18 ]; after separation by 8% to 12% polyacrylamide gel electrophoresis, the following antibodies were used to confirm protein expression: primary antibody against Bcl-2 (sc-7382; Santa Cruz, Germany), c- myc (sc-764; Santa Cruz), BARF1 (anti-Pep 2A serum, a polyclonal rabbit antiserum prepared against a synthetic peptide corresponding to a presumed epitope, amino acids (aa) 172 to 180 [NGGVMKEKD], of the BARF1 protein), Ras (sc-29; Santa Cruz), SV40 Tantigen (sc-148; Santa Cruz; N-terminal epitope mapping within residues 1 to 82 of SV40 large Tantigen for detection of 94-kDa SV40 LT antigen and 21-kDa SV40 ST antigen), LMP1 (BD Biosciences Pharmingen, San Diego, CA), h-TERT (SC-7212; Santa-Cruz), and tubulin (sc-5286; Santa Cruz).

Techniques: Expressing, Transfection, Injection, Produced, Recombinant, Positive Control, Cell Culture, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Clone Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Synergism of BARF1 with Ras Induces Malignant Transformation in Primary Primate Epithelial Cells and Human Nasopharyngeal Epithelial Cells 1

doi:

Figure Lengend Snippet: Colony Formation in Soft Agar and Tumor Formation in Nude Mice.

Article Snippet: Western Blot Analysis Immunoblot analysis was performed as previously described [ 18 ]; after separation by 8% to 12% polyacrylamide gel electrophoresis, the following antibodies were used to confirm protein expression: primary antibody against Bcl-2 (sc-7382; Santa Cruz, Germany), c- myc (sc-764; Santa Cruz), BARF1 (anti-Pep 2A serum, a polyclonal rabbit antiserum prepared against a synthetic peptide corresponding to a presumed epitope, amino acids (aa) 172 to 180 [NGGVMKEKD], of the BARF1 protein), Ras (sc-29; Santa Cruz), SV40 Tantigen (sc-148; Santa Cruz; N-terminal epitope mapping within residues 1 to 82 of SV40 large Tantigen for detection of 94-kDa SV40 LT antigen and 21-kDa SV40 ST antigen), LMP1 (BD Biosciences Pharmingen, San Diego, CA), h-TERT (SC-7212; Santa-Cruz), and tubulin (sc-5286; Santa Cruz).

Techniques: Plasmid Preparation

Journal: FEBS letters

Article Title: Inactivation of a MAPK-like protein kinase and activation of a MBP kinase in germinating barley embryos.

doi: 10.1016/s0014-5793(00)02112-8

Figure Lengend Snippet: Fig. 1. Activity of MBP phosphorylating kinases and expression of MAP kinases in barley embryos. (A) In-gel MBP kinase assay of embryo extracts. Intact barley grains were imbibed in water and embryos were dissected at designated times. 30 Wg of protein was loaded on the gel. (B) Control gel without MBP. Samples are identical to samples loaded in (A). (C) Immunoprecipitation followed by in-gel kinase assay. Protein extracts of dry embryos and 8 h and 48 h imbibed embryos (200 Wg) were used for immunoprecipitation with anti-ERK1 antibody. Immuno- complexes were analyzed for MBP phosphorylation by in-gel kinase assay. Lanes 1^3: control IP using only protein G, no antibody. Lanes 4^ 6: immunoprecipitations with protein G and anti-ERK1 antibody. Lanes 7^9: total extracts 20 Wg. One representative example of three inde- pendent experiments is presented. (D) Western analysis of barley embryo extracts with anti-ERK1 antibody. 20 Wg of protein of each sample was loaded.

Article Snippet: For immunoprecipitation (basically according to Knetsch et al. [8]), 200 Wg of total protein extract was added to 10 Wl anti-ERK1 antibody (K-23, rabbit polyclonal sc-94, raised against a peptide which corresponds to amino acids 305^327 mapping within subdomain XI of ERK1-encoded MAP kinase p44 of rat origin, Santa Cruz Biotechnology) that was precoupled to 20 Wl protein G-Sepharose 4B-fast £ow (Pharmacia).

Techniques: Activity Assay, Expressing, Kinase Assay, Control, Immunoprecipitation, Phospho-proteomics, Western Blot