Journal: Egyptian Journal of Medical Human Genetics

Article Title: Ankaferd Blood Stopper induces apoptosis and regulates PAR1 and EPCR expression in human leukemia cells

doi: 10.1016/j.ejmhg.2014.10.001

Figure Lengend Snippet: Figure 3 Apoptosis detection by Annexin V staining in K-562 and Jurkat cells after ABS treatment.

Article Snippet: Apoptosis detection was performed using Annexin V apoptosis detection kit (sc-4252 AK) (Santa Cruz Biotechnology, Inc.) on live cells according to the manufacturer’s recommendations.

Techniques: Staining

Journal: Frontiers in cell and developmental biology

Article Title: RNA-binding is an ancient trait of the Annexin family.

doi: 10.3389/fcell.2023.1161588

Figure Lengend Snippet: FIGURE 1 AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, AnxA10, AnxA11 and AnxA13 present in the cytoskeleton fraction (Panel A) are associated with non-polysomal mRNP complexes (Panel B) of PC12 cells. Panel (A) 30 µg of the cytoskeletal fraction (lane 1) and cytoskeleton-bound polysomes (lane 2) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Panel (B) samples prepared from oligo (dT)-bound mRNP complexes from the cytoskeletal fraction [supernatant after centrifugation for 2 h 100,000 g above a 1 M (35%) sucrose cushion] (lanes 3 and 4), without (lane 3) or with RNase (lane 4) treatment, as indicated above the Western blots, were subjected to similar analysis. Theblots were probed with antibodies against the different Anxs and against PABP1 as a marker for poly(A)-containing mRNAs, as indicated. Antibodies against the ribosomal subunit S6 were used to inform of the distribution of ribosomes. In addition, the blots were probed with antibodies against early endosomes (EEA1), late endosomes (Rab7) and recycling endosomes (Rab11). SPC25 and LAMP1 were not detectable in any of the fractions (results not shown). Visualization of the immunoreactive protein bands was performed using the ChemiDocTM XRS+ molecular imager after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL)-reagent. The blots shown are representative for results from three experiments.

Article Snippet: The membranes were probed against AnxA1 (HPA011272, Sigma-Aldrich, Saint-Louis, United States; 1:1,000), AnxA2 (610069; BD Biosciences, Franklin Lakes, United States; 1:1,000), AnxA4 (PA5-82296, Invitrogen, Thermo Fisher Scientific, Waltham, United States; 1:1,000), AnxA5 (MA5-35789, Invitrogen, Thermo Fisher Scientific, Waltham, United States; 1:1,000), AnxA6 (NBP1-90149, Novus Biologicals LTD., Bristol, UK; 1:1,000), AnxA7 (sc-17815, Santa Cruz Biotechnology, Dallas, United States; 1:1,000), AnxA10 (ab213656, Abcam, Cambridge, UK; 1:1,000), AnxA11 (sc-9322, Santa Cruz Biotechnology, Dallas, United States; 1:1,000), AnxA13 (PA5-109395, Invitrogen, Thermo Fisher Scientific, Waltham, United States; 1:1,000)), Rab7 (R4779, Sigma/Merck,; 1:1,000), Rab11 (610657, BD Transduction Lab/Thermo Fisher Scientific, Waltham, United States; 1:1,000), EEA1 (610457, BD Transduction Lab/Thermo Fisher Scientific, Waltham, United States; 1:1,000), S6 (710405; Thermo Fisher Scientific; Waltham, United States; 1:1,000), and PABP1 (4992S; Cell Signaling Technology, Danvers, United States; 1:1,000) primary antibodies.

Techniques: SDS Page, Centrifugation, Western Blot, Marker, Incubation

Journal: Frontiers in cell and developmental biology

Article Title: RNA-binding is an ancient trait of the Annexin family.

doi: 10.3389/fcell.2023.1161588

Figure Lengend Snippet: FIGURE 3 UV-crosslinking competition experiments employing rat Anxs and radiolabeled [α32P]-rUTP c-myc or anxA2 3′UTRs. 2 μM purified rat Anxs (except 0.4 µM of AnxA7; 0.3 µM of AnxA9 and 0.5 µM of AnxA11) were UV-crosslinked in the absence of RNA (lanes 1 and 5). 100,000 cpm of radiolabeled anxA2 3′UTR (7 fmoles) or c-myc 3′UTR (6 fmoles) were UV-crosslinked to purified rat Anxs in the absence (lanes 2 and 6) or presence of 25x (lanes 3 and 7), or 50x (lanes 4 and 8) molar excess of the corresponding unlabeled transcript. 2 μM BSA and mutant AnxA2 served as negative controls. After UV-crosslinking and RNase treatment, the samples were subjected to 4%–15% SDS-PAGE and the proteins were stained with Coomassie Brilliant Blue (lanes 1–4), whereafter the gels were dried. The [α32P]-rUTP-labeled RNA covalently bound to the respective Anxs, as indicated, was visualized using screens and phosphor-imaging following an overnight (c-myc 3′UTR) or 6 h (anxA2 3′UTR) exposure (lanes 5–8). PageRuler prestained protein ladder (from top to bottom: 100 kDa, 70 kDa (the most prominent band), 55 kDa, 40 kDa, 35 kDa and 25 kDa) are shown to the left of the AnxA1, AnxA3, AnxA5, AnxA7, AnxA8, AnxA10, Δ188AnxA11 and BSA samples. The representative images are from two experiments performed with competition while RNA-Anx binding was performed four times without competition.

Article Snippet: The membranes were probed against AnxA1 (HPA011272, Sigma-Aldrich, Saint-Louis, United States; 1:1,000), AnxA2 (610069; BD Biosciences, Franklin Lakes, United States; 1:1,000), AnxA4 (PA5-82296, Invitrogen, Thermo Fisher Scientific, Waltham, United States; 1:1,000), AnxA5 (MA5-35789, Invitrogen, Thermo Fisher Scientific, Waltham, United States; 1:1,000), AnxA6 (NBP1-90149, Novus Biologicals LTD., Bristol, UK; 1:1,000), AnxA7 (sc-17815, Santa Cruz Biotechnology, Dallas, United States; 1:1,000), AnxA10 (ab213656, Abcam, Cambridge, UK; 1:1,000), AnxA11 (sc-9322, Santa Cruz Biotechnology, Dallas, United States; 1:1,000), AnxA13 (PA5-109395, Invitrogen, Thermo Fisher Scientific, Waltham, United States; 1:1,000)), Rab7 (R4779, Sigma/Merck,; 1:1,000), Rab11 (610657, BD Transduction Lab/Thermo Fisher Scientific, Waltham, United States; 1:1,000), EEA1 (610457, BD Transduction Lab/Thermo Fisher Scientific, Waltham, United States; 1:1,000), S6 (710405; Thermo Fisher Scientific; Waltham, United States; 1:1,000), and PABP1 (4992S; Cell Signaling Technology, Danvers, United States; 1:1,000) primary antibodies.

Techniques: Mutagenesis, SDS Page, Staining, Labeling, Imaging, Binding Assay