Fig 2: Western-blot analysis to compare circulating antibodies generated against autologous tumor antigens pre- and post-CRAd administration. For each patient, autologous blood plasma was used as primary antibody and goat anti-dog IgG-HRP antibody was used as secondary antibody. The blots were scanned using C-Digit Blot Scanner (LICOR).

Fig 3: Expression of C3 mRNA and C3, C3b, and iC3b proteins in the decidual and placental tissues.(A) Levels of C3 mRNAs obtained from P11, P15, or P19 decidua (solid bar) and P11, P15, or P19 placenta (white bar) (n = 3 mice each day). Actb mRNA was used as an internal control for RNA integrity. Values represent mean ± SEM from three animals with duplicate samples. Different superscripts indicate statistically significant differences in C3 mRNA levels (p < 0.05) when compared to that of P11. (B) Western blot analysis of C3, C3b, or iC3b expression in P11, P15, or P19 decidual and P11, P15, or P19 placental tissues. ACTB was used as an internal control. A representative data from three independent experiments containing protein samples from three different animals is shown. (C) Densitometric quantitation of C3, C3b, and iC3b on Western blot analysis. The analysis was done using LI-COR C-DiGit Blot Scanner and the relative density of the blots was determined by normalization against ACTB levels. Values represent mean ± SEM from three animals with duplicate samples. Different superscripts (small letters, decidua) or capital letters (placenta) indicate statistically significant differences in mRNA levels (p < 0.05) within the tissues when compared to that of P11.

Fig 4: UTAG expression in mammalian cells.(A) UTAG localization to sites of sumoylation in mammalian cells. mCherry-tagged KmUTAG and pEYFP-SUMO1 were transfected into mammalian cells that were grown on cover slips. Cells were then fixed, stained with DAPI, and visualized using a confocal microscope. An overlay of DAPI, mCherry, and YFP images is shown in the lower right corner (merge). Expression of kmUTAG did not alter the distribution or localization of YFP-SUMO (B) Using UTAG to pulldown SUMO-modified proteins from mammalian cell extracts. Whole cell extracts (WCE) of 786–0 renal carcinoma cells transfected with YFP-SUMO1, control GFP or untransfected (none). Pulldowns using recombinant KmUTAG on the right. Note high molecular weight YFP-SUMO1 conjugates in the pulldown of YFP-SUMO1. The graphic representation to the right indicates that KmUTAG was immobilized on amylose beads (circle) and that YFP-SUMO conjugates were pulled down. (C) 5ug KmUTAG, SCUTAG or no protein (mock) were added to 100ul diluted cell extracts (see Materials & methods) and nutated in the presence of nickel beads for 1 hour at 25°C. Eluted proteins were run on SDS-PAGE, western blotted with anti SUMO1 antibody, and quantitated using a c-DIGIT scanner (Li-Cor Biosciences). 20% of input whole cell extract (WCE) probed with the anti SUMO1 antibody is shown for comparison.

Fig 5: Local anesthetics induce autophagy in tooth pulp cells. (a) Phase-contrast microscopy images of drug-treated and control cells. (b and c) Immunofluorescence analysis of LAMP-1 and LC3 (red) expression in control cells and cells treated with 2 mM anesthetic drug. (d) Western blot (left panel) analysis of LC3 expression in two primary dental pulp cell lines with and without drug treatment. Note that LC3 is expressed as two isoforms with molecular weights 17 kDa (LC3-II) and 19 kDa (LC3-I). ß-Actin was used as a loading control. Signal quantification (right panel) was performed on C-DiGit Blot scanner acquired gel images using Image Studio 4.0 software (LI-COR Biosciences UK Ltd, Cambridge, UK). Relative signal was calculated as LC3II (17 kDa) versus LC3I (19 kDa) then normalized against ß-Actin signal. (e) GFP–LC3 fusion protein expression (green) in control cells and cells treated with 0.5 mM anesthetic drug. Cells were counterstained for Phalloidin (red). Cell nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI). Additional analysis can be found in Supplementary Figure 2. Scale bars, 20 µm. UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Scale bars, 10 µm.