Journal: International journal of oncology

Article Title: An asymmetrically dimethylarginated nuclear 90 kDa protein (p90aDMA) induced by interleukin (IL)-2, IL-4 or IL-6 in the tumor microenvironment is selectively degraded by autophagy.

doi: 10.3892/ijo.2016.3450

Figure Lengend Snippet: Figure 5. The p90aDMA and p70aDMA proteins accumulate in the nucleus of cancer cells and degrade in response to cellular stresses. (A) Nuclear and cytoplasmic extracts were prepared after MDA-MB‑231 cells were cultured with IL-2 or IL-6 as indicated. Fractions were analyzed by western blot analysis using antibody to ASYM24. TBP was probed as a loading control. (B and C) MDA-MB‑231 cells or HeLa cells were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. (D) HeLa cells were cultured in HBSS medium for 24 h to induce autophagy or left in the complete culture medium. The cells were harvested and nuclear and cytoplasmic extracts were prepared for western blot analysis using antibodies to LC3 and ASYM24, respectively. Fractionation efficiency was determined by localization of β-actin (cytoplasm) and TBP (nucleus).

Article Snippet: Target Source Host Dilution Catalog SYM11 Merck Millipore, MA, USA Rabbit 1:1,000 07-413 ASYM24 Merck Millipore, MA, USA Rabbit 1:500 07-414 LC3B Cell Signaling Technology, Inc. (CST), MA, USA Rabbit 1:1,000 #3868 ATG5 Cell Signaling Technology, Inc. (CST), MA, USA Rabbit 1:1,000 #12994 TBP Proteintech, Wu Han, China Rabbit 1:500 22006-1-AP β-actin Zen Bioscience, Cheng Du, China Mouse 1:20,000 70068 that were significantly reduced regardless of the interleukin stimulation (Fig. 2).

Techniques: Cell Culture, Western Blot, Control, Fractionation

Journal: Cell Death & Disease

Article Title: Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis

doi: 10.1038/s41419-021-04172-y

Figure Lengend Snippet: A β-Catenin (a marker of adherens junctions, green) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. B ZO2 (a marker of tight junctions, green) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. C TEX14 (a marker of a marker of testicular intercellular junctions, green) and β-actin (red) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. D Vimentin (a marker for Sertoli apical extensions, green) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. Nuclei are stained with DAPI.

Article Snippet: PPP6C antibody (rabbit, A300-844A; Bethyl Laboratories, Inc.); SYCP3 antibody (rabbit, NB300-231; Novus Biologicals); α-tubulin antibody (rabbit, 2144; Cell Signaling Technology, Inc.); β-actin antibody (mouse, 3700; Cell Signaling Technology, Inc.); Phospho-β-Catenin (Ser552) (D8E11) antibody (rabbit, 5651; Cell Signaling Technology, Inc.); SYCP3 antibody (mouse, sc-74569; Santa Cruz); γH2AX antibody (rabbit, 9718; Cell Signaling Technology, Inc.); MVH antibody (mouse, ab27591; abcam); SYCP1 antibody (rabbit, ab15090; abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); β-catenin antibody (rabbit, 51067-1-AP, Proteintech); ZO2 antibody (rabbit, 18900-1-AP, Proteintech); TEX14 antibody (rabbit, 18351-1-AP, Proteintech); Vimentin antibody (rabbit, 10366-1-AP, Proteintech); HA-Tag mab (mouse, AE008; ABclonal); c-Myc antibody (mouse, m4439; sigma); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo).

Techniques: Marker, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Specnuezhenide Decreases Interleukin-1β-Induced Inflammation in Rat Chondrocytes and Reduces Joint Destruction in Osteoarthritic Rats

doi: 10.3389/fphar.2018.00700

Figure Lengend Snippet: Effect of SPN on IL-1β-induced nuclear translocation of NF-κB p65 and β-catenin. Nuclear and cytoplasmic extraction reagents were used to prepare nuclear (A) and cytoplasmic (B) extracts. Then, protein levels of β-catenin, NF-κB p65, GAPDH in the cytoplasm, and β-catenin, NF-κB p65, and TBP in the nucleus were assessed by Western blotting. GAPDH was used as an endogenous control in the cytoplasm, whereas TBP worked as an endogenous control in the nucleus ( n = 3 per group). (C) Chondrocytes were pretreated with SPN for 1 h, and then stimulated with IL-1β for 6 h for NF-κb-Luc activity analysis, or 12 h for TCF-LEF RE activity analysis ( n = 5 per group). Significance was calculated by one-way ANOVA with post hoc Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗∗ p < 0.001 versus the IL-1β+SPN (0 μM) group. # p < 0.05, ## p < 0.01, ### p < 0.001 versus the NC group.

Article Snippet: In the present study, Ab against MMP-3 (rabbit mAb, Abcam, ab52915), MMP-9 (rabbit mAb, Abcam, ab76003), IL-6 (10E5, mouse mAb, Santa Cruz, sc-57315), iNOS (rabbit mAb, Abcam, ab3523), COX2 (D5H5, rabbit mAb, Cell Signaling, #12282), collagen 2 (rabbit mAb, Abcam, ab188570), sox9 (rabbit mAb, Abcam, ab185966), β-catenin (D10A8, rabbit mAb, Cell Signaling, #8480p), non-phospho (active) β-catenin (Ser45) (D2U8Y, rabbit mAb, Cell Signaling, #19807S), NF-κB p65 (C22B4, rabbit mAb, Cell Signaling, #4764S), phosphor-NF-κB p65 (Ser536) (rabbit Ab, Cell Signaling, #3031), β-actin (mouse mAb, Abcam, ab8226), GAPDH (rabbit mAb, Cell Signaling, #5174), and TBP (rabbit Ab, Bosterbio, BA3586-2) were used.

Techniques: Translocation Assay, Extraction, Western Blot, Control, Activity Assay