Journal: Scientific Reports

Article Title: Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins

doi: 10.1038/s41598-018-34473-w

Figure Lengend Snippet: Western blot and ELISA analysis of HMBS and ATP2C1. ( a ) Cell lysates were applied to SDS-PAGE gels under reducing conditions. HMBS and ATP2C1 protein were detected in platelets using a polyclonal antibody. Incubation of platelets with E. coli K12 and E. coli O18:K1 in 1:5 or 1:10 platelet-bacteria ratios converted HMBS 47 kDa form to a 40 kDa protein. ATP2C1 was not affected. GAPDH was used as a loading control. ( b ) The releasates of the platelet-bacteria mix were collected after centrifugation (500 g, 10 minutes without break). HMBS, ATP2C1 and LRCH4 levels were measured by ELISA. Data represents the mean of three independent experiments (n = 3). ATP2C1 was detectable in platelet supernatants, while HMBS and LRCH4 proteins were either not released from platelets or in concentrations below the detection level of the ELISA (data not shown). BDL, below detection limit. Western blot results are representative image of three replications. The same exposure was applied equally across the entire image. The original pictures of the full-length western blots can be found in Supplementary Fig. .

Article Snippet: The blots were incubated overnight with primary anti-human antibodies (rabbit polyclonal ATP2C1 (1:750), LRCH4 (1:500), HMBS (1:1000) or GAPDH (1:600) from Atlas Antibodies, Bromma, Sweden).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation, Bacteria, Control, Centrifugation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Acupuncture Alters Expression of Insulin Signaling Related Molecules and Improves Insulin Resistance in OLETF Rats

doi: 10.1155/2016/9651592

Figure Lengend Snippet: Effects of acupuncture on protein expressions of PI3K-p85, phospho-PKC ζ / λ , and GLUT4 in skeletal muscle of OLETF rats. Protein expression was determined by Western blot. GAPDH was used as an internal control. Data are shown as mean ± SD ( n = 8 each group). ∗ P < 0.05 and ∗∗ P < 0.01 versus SD group; △△ P < 0.01 versus OLETF group.

Article Snippet: Nonspecific binding sites were blocked with 5% milk powder diluted in TBS with 0.05% Tween 20 (TBST) for 60 min. Proteins were detected using the following antibodies: rabbit polyclonal antibody for PI3K-p85 (diluted 1 : 3000; Bios; bs-0128R), rabbit polyclonal antibody for phospho-PKC ζ / λ (diluted 1 : 3000; CST; #9378), rabbit polyclonal antibody for GLUT4 (diluted 1 : 3000; Boster; BA1626), and mouse monoclonal antibody for GAPDH (diluted 1 : 3000; Boster; BM1623).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Physiology

Article Title: Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

doi: 10.3389/fphys.2017.00336

Figure Lengend Snippet: Western blot analyses of PMCA1-4, and NCKX3 in secretory-stage and maturation-stage rat enamel organs. (A) Western blot analysis for PMCA1 (Ai) , PMCA2 (Aii) , PMCA3 (Aiii) and PMCA4 (Aiv) . Samples are secretory-stage enamel organ cells (S), maturation-stage enamel organ cells (M), brain tissue (B) and heart tissue (H). Brain and heart samples are shown for comparison, as all PMCAs are highly expressed in brain and at lower levels in the heart (Brini and Carafoli, ; Brini et al., ). Molecular weight markers are indicated at left. The expected molecular weights for PMCA1, PMCA2, PMCA3, and PMCA4 are ~130, 133, 123, and 129 kDa, respectively. The bands are seen for PMCA1, PMCA3, and PMCA4 (boxed and arrow). No expression of PMCA2 is evident. GAPDH is used here as a loading control. (B) Western blot analysis of NCKX3. The expected molecular weight for NCKX3 is ~60 kDa. NCKX3 is expressed in all 4 tissue samples tested, with similar expression noted in both secretory-stage and maturation-stage enamel organ cells, and brain tissue. Relatively higher levels of NCKX3 expression can be appreciated in heart tissue. Amelx and Actc are used as controls as expression is highest in secretory-stage ameloblasts (Lacruz et al., , ) and heart tissue (Hamada et al., ) respectively. GAPDH is used here as a loading control.

Article Snippet: Antibodies against PMCA1 (AbCam, Cambridge, MA, USA; catalog #ab190355), PMCA2 (ab3529), PMCA3 (ab3530), PMCA4 (ab2783), NCKX3 (St. John's Laboratory, London, UK, catalog #STJ94358), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, catalog #sc-32233), amelogenin (ThermoFisher Scientific, catalog #PA5-31286), and cardiac muscle actin (ACTC1) (GeneTex Inc., Irvine, CA, catalog #GTX101876) were used at dilutions of 1:500, 1:2,000, 1:300, 1:5,000, 1:500, 1:500, 1:3,000, and 1:500, respectively in 5% milk in TBST.

Techniques: Western Blot, Comparison, Molecular Weight, Expressing, Control