Journal: Nucleic Acids Research

Article Title: HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

doi: 10.1093/nar/gkq412

Figure Lengend Snippet: Effect of HLJ1 on activation of JNK and caspase-3 after UV irradiation. ( A ) Ectopic expression of HLJ1 enhances JNK activity after UV irradiation. Mock (mock-1 and mock-2) and HLJ1 transfectants (HLJ1-1 and HLJ1-2) were irradiated with 50 J/m 2 of UV, followed by recovery for 15 or 60 min. Activated and total JNKs were detected by western blotting with antibodies specific for JNK and p-JNK, respectively. ( B ) Enforced expression of HLJ1 enhances caspase-3 and -9 activities. Mock and HLJ1 transfectants were exposed to 50 J/m 2 of UV, and allowed to recover for 12 or 24 h. Cell extracts were analyzed for caspase-3, -9 and PARP by western blotting with anti-caspase-3, anti-caspase-9 and anti-PARP antibodies. β-tubulin was used as a control for protein loading.

Article Snippet: The primary antibodies used for western blot analyses included monoclonal mouse anti-HLJ1 (made in-house), polyclonal goat anti-Hsc70 antibody (sc-1059, Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse anti-Hsp70 antibody (sc-24, Santa Cruz Biotechnology), polyclonal rabbit anti-SAPK/JNK antibody (Cat. 9252, Cell Signaling Technology, Beverly, MA, USA), monoclonal mouse anti-phospho-SAPK/JNK (Thr183/Tyr185) antibody (Cat. 9255, Cell Signaling Technology), polyclonal rabbit anti-caspase-3 antibody (Cat. AB1899, Millipore), monoclonal mouse anti-caspase-9 antibody (Cat. 551246, BD Biosciences), polyclonal rabbit anti-PARP antibody (Cat. 5242, Cell Signaling Technology), and monoclonal mouse anti-β-tubulin (Cat. 05-661, Millipore) used as a loading control.

Techniques: Activation Assay, Irradiation, Expressing, Activity Assay, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

doi: 10.1093/nar/gkq412

Figure Lengend Snippet: HLJ1 reduction by UV irradiation. ( A ) HLJ1 is decreased following UV exposure in a dose-dependent manner. CL1-0 cells were irradiated with 10, 20 or 50 J/m 2 of UV, and allowed to recover for 6 h. Expression of HLJ1, Hsc70 and Hsp70 was detected by western blot analysis. ( B ) HLJ1 is decreased after UV exposure in a time-dependent manner. CL1-0 cells were irradiated with 10 J/m 2 of UV, and allowed to recover for 3, 6, 9, 12 or 24 h. Cell extracts were analyzed by western blot with anti-HLJ1, anti-Hsc70, anti-PARP and anti-caspase-3 antibodies. ( C ) Ectopically expressed HLJ1 is decreased after UV exposure. Mock- and HLJ1-transfected cells derived from CL1-5 were exposed to 50 J/m 2 of UV irradiation, followed by recovery for 12 h. HLJ1 was detected by western blot analysis using an anti-HLJ1 antibody. β-tubulin was used as the internal control.

Article Snippet: The primary antibodies used for western blot analyses included monoclonal mouse anti-HLJ1 (made in-house), polyclonal goat anti-Hsc70 antibody (sc-1059, Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse anti-Hsp70 antibody (sc-24, Santa Cruz Biotechnology), polyclonal rabbit anti-SAPK/JNK antibody (Cat. 9252, Cell Signaling Technology, Beverly, MA, USA), monoclonal mouse anti-phospho-SAPK/JNK (Thr183/Tyr185) antibody (Cat. 9255, Cell Signaling Technology), polyclonal rabbit anti-caspase-3 antibody (Cat. AB1899, Millipore), monoclonal mouse anti-caspase-9 antibody (Cat. 551246, BD Biosciences), polyclonal rabbit anti-PARP antibody (Cat. 5242, Cell Signaling Technology), and monoclonal mouse anti-β-tubulin (Cat. 05-661, Millipore) used as a loading control.

Techniques: Irradiation, Expressing, Western Blot, Transfection, Derivative Assay, Control

Journal: Nucleic Acids Research

Article Title: HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

doi: 10.1093/nar/gkq412

Figure Lengend Snippet: Caspase-dependent HLJ1 reduction in apoptotic cells. ( A ) Decreased expression of HLJ1 was pronounced upon inhibition of protein synthesis. CL1-0 cells were pre-incubated with 10 μg/ml of CHX for an hour, and exposed to 10 J/m 2 of UV. After recovery for 6 h, HLJ1 expression was analyzed by western blot using anti-HLJ1 antibody (left) and real-time RT–PCR (right). ( B ) The majority of detached cells induced by UV irradiation were apoptotic. CL1-0 cells were irradiated with 10 J/m 2 of UV, and allowed to recover for 24 h. Based on adherence, total cells including detached and adherent, adherent cells and detached cells, were harvested. The DNA content of cells was analyzed by flow cytometry, as described in ‘Materials and Methods’ section. ( C ) HLJ1 was absent in detached cells. CL1-0 and HeLa cells were treated with 10 J/m 2 of UV irradiation, and allowed to recover for 24 h. Total cells (T), adherent cells (A) and detached cells (D) were collected for western blot analysis with anti-HLJ1 and anti-Hsc70 antibodies. β-Tubulin was applied as the internal control. Results are representative of three independent experiments. ( D ) Caspase inhibitors attenuated UV-induced cell apoptosis. CL1-0 cells were pre-incubated for 3 h with 20 μM zVAD-fmk or 30 μM DEVD-fmk, followed by exposure to 10 J/m 2 of UV irradiation. After recovery for 6 h, the apoptotic cell content was determined based on the sub-G1 proportion using a flow cytometer. Data are presented as mean ± S.D. ( n = 3). ( E ) Caspase inhibitors blocked UV-induced HLJ1 degradation. Western blots were performed with antibodies against HLJ1, PARP or β-tubulin. PARP is a caspase-3 substrate, while β-tubulin is the internal control.

Article Snippet: The primary antibodies used for western blot analyses included monoclonal mouse anti-HLJ1 (made in-house), polyclonal goat anti-Hsc70 antibody (sc-1059, Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse anti-Hsp70 antibody (sc-24, Santa Cruz Biotechnology), polyclonal rabbit anti-SAPK/JNK antibody (Cat. 9252, Cell Signaling Technology, Beverly, MA, USA), monoclonal mouse anti-phospho-SAPK/JNK (Thr183/Tyr185) antibody (Cat. 9255, Cell Signaling Technology), polyclonal rabbit anti-caspase-3 antibody (Cat. AB1899, Millipore), monoclonal mouse anti-caspase-9 antibody (Cat. 551246, BD Biosciences), polyclonal rabbit anti-PARP antibody (Cat. 5242, Cell Signaling Technology), and monoclonal mouse anti-β-tubulin (Cat. 05-661, Millipore) used as a loading control.

Techniques: Expressing, Inhibition, Incubation, Western Blot, Quantitative RT-PCR, Irradiation, Flow Cytometry, Control

Journal: Nucleic Acids Research

Article Title: HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

doi: 10.1093/nar/gkq412

Figure Lengend Snippet: Effect of HLJ1 cleavage on UV-induced apoptosis. ( A ) HLJ1-D128A mutant is resistant to UV-induced apoptosis. After UV irradiation, the sub-G1 percentages of vector-transfected control cells (Mock), HLJ1-transfected cells (HLJ1) and mutant HLJ1-transfected cells (D128A-mixed clone and D128A-single clone) were detected by flow cytometry. Data are presented as mean ± S.D. of three independent experiments. ( B ) UV-induced JNK activation is defective in mutant HLJ1 transfectant. Wild-type HLJ1 and mutant HLJ1 (D128A) transfectants were irradiated by UV light followed by incubation at 37°C for 15 or 30 min. JNK activity was detected by western blotting with an anti-p-JNK antibody. α-Actinin served as the loading control. The expression of V5-tagged HLJ1 in transfectant was detected with an anti-V5 tag antibody. ( C ) Rescue of the caspase pathway by expression of HLJ1-D128A mutant protein. Mock, HLJ1 and mutant HLJ1 transfectants were exposed to 50 J/m 2 of UV, and recovered for 24 and 48 h. Cell extracts were analyzed for V5-tagged HLJ1, caspase-9 and PARP by western blotting with the indicated antibodies. GAPDH was used as a control for protein loading.

Article Snippet: The primary antibodies used for western blot analyses included monoclonal mouse anti-HLJ1 (made in-house), polyclonal goat anti-Hsc70 antibody (sc-1059, Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse anti-Hsp70 antibody (sc-24, Santa Cruz Biotechnology), polyclonal rabbit anti-SAPK/JNK antibody (Cat. 9252, Cell Signaling Technology, Beverly, MA, USA), monoclonal mouse anti-phospho-SAPK/JNK (Thr183/Tyr185) antibody (Cat. 9255, Cell Signaling Technology), polyclonal rabbit anti-caspase-3 antibody (Cat. AB1899, Millipore), monoclonal mouse anti-caspase-9 antibody (Cat. 551246, BD Biosciences), polyclonal rabbit anti-PARP antibody (Cat. 5242, Cell Signaling Technology), and monoclonal mouse anti-β-tubulin (Cat. 05-661, Millipore) used as a loading control.

Techniques: Mutagenesis, Irradiation, Plasmid Preparation, Transfection, Control, Flow Cytometry, Activation Assay, Incubation, Activity Assay, Western Blot, Expressing

Journal: Pharmaceuticals

Article Title: Phytochemical Composition and Skin-Friendly Activities of the Ethyl Acetate Fraction in Ophioglossum vulgatum Linn., an In Vitro Study

doi: 10.3390/ph18030345

Figure Lengend Snippet: List of antibodies for WB.

Article Snippet: NRF2 , 1:1000 , BOSTER , A00078-1.

Techniques: