Journal: Molecular medicine reports

Article Title: A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

doi: 10.3892/mmr.2015.4059

Figure Lengend Snippet: Figure 4. Overexpression of ECRG4 induced apoptosis in laryngeal cancer cells through regulation of the expression of apoptosis‑related factors. (A) Examination of apoptosis by flow cytometric analysis. The figure shows the representative results of several repeated experiments. (B) Examination of apoptosis by Hoechst staining. Apoptotic cells exhibited intensive chromatin condensation and dense, strongly Hoechst stained nuclei (magnification, x400). (C) Examination of the expression of Bax, Bcl‑2, cleaved‑caspase‑3 and cleaved‑PARP by western blot analysis. Grayscale analysis was performed using β‑actin as the internal control. Experimental data are expressed as the mean ± standard deviation. Compared with the pcDNA3.1 group, **P<0.01. ECRG4, human esophageal cancer-related gene 4; Bax, Bcl‑2‑associated X protein; Bcl-2, anti‑B‑cell lymphoma 2; PARP, poly ADP‑ribose polymerase.

Article Snippet: The membranes were incubated first with the following primary antibodies: Rabbit anti-human anti-ECRG4 polyclonal antibody (1:200; cat. no. sc-135139; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human anti-cleaved-caspase-3 polyclonal antibody (1:500; cat. no. bs-0081R; Bioss, Beijing, China), rabbit anti-human anti-cleaved-poly ADP-ribose polymerase (PARP) polyclonal antibody (1:200; cat. no. sc-23461-R; Santa Cruz Biotechnology, Inc.), rabbit anti-human anti-Bcl-2-associated X protein (Bax) polyclonal antibody (1:400; cat. no. BA0315; Boster, Wuhan, China) and rabbit anti-human anti-B-cell lymphoma 2 (Bcl-2) polyclonal antibody (1:400; cat. no. BA0412; Boster) at 4 ̊C overnight.

Techniques: Over Expression, Expressing, Staining, Western Blot, Control, Standard Deviation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: UFL1 and C53 interact with γTuRC proteins. ( A ) Immunoprecipitation experiments. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized Abs specific to UFL1 301–389 , C53, γ-tubulin (γ-Tb), or GCP2. The blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, GCP4, or calcineurin (Calcin.; negative control). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ). ( B ) Isotype controls. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized rabbit Ab to myosin or mouse mAb to MAP2 (IgG2b). Blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, or GCP4. Load ( lane 1 ), immobilized Abs not incubated with cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and carriers without Abs incubated with cell extracts ( lane 4 ). ( C ) The size distribution of UFL1, C53, γ-tubulin (γ-Tb), GCP2, and actin in U2OS whole-cell extracts fractionated on the Superose 6 column. The calibration standards (in kDa) are indicated on the top. The numbers at the bottom denote individual fractions. ( D ) Pooled fractions (Nos. 15–16) and (Nos. 23–24) from fractionation shown in panel ( C ) were precipitated with Ab to γ-tubulin. The blots were probed with Abs to UFL1, C53, and γ-tubulin (γ-Tb). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ).

Article Snippet: Rabbit Ab to calcineurin (2614) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Negative Control, Incubation, Fractionation