Microcon ym 3

Apoplastic proteins were extracted as previously described by Hon et al. [17 (link)], with slight modifications. Leaf blades (50 g) of non-acclimated and cold-acclimated wheat plants were harvested and cut into 3 cm segments. These segments were gathered and tied, and the cut surface was washed with deionized water. Subsequently, the leaf segments were soaked in an extraction buffer (20 mM ascorbic acid, 20 mM CaCl₂, pH 3.0) and vacuum infiltrated for 30 min. The leaf segments were then surface-dried with a paper towel and then placed in a plastic syringe barrel (10 mL). Afterwards, the syringe barrel was inserted into a 15 mL conical tube. Apoplastic fluids were recovered by centrifugation at 2000× g for 10 min using a swing out rotor. The fluid collected at the bottom of the tube was subsequently concentrated with a Microcon YM-3 (Millipore, Burlington, MA, USA). Protein concentration was estimated by using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and BSA was used as a standard.

According to the experimental requirements, NB cells and iBMSC were inoculated into the upper chamber and the lower chamber respectively and co-cultured for 24–48 h. Thereafter, the medium was changed to FBS-free medium, and the cells were cultured for another 48 h. NB cells or iBMSC cultured separately under the same conditions were used as controls. For the CM from NB cells treated with LPC or other reagents, the culture plate shall be cleaned with serum-free medium and replaced with new medium for another round of culture after administration. The medium was then collected and centrifuged at 1,000 × g at 4°C for 10 min and the supernatant was concentrated at 12,500 g for 30 min in a Microcon YM-3 centrifugal filter device (Millipore Co., Bedford, MA).

Heparan sulfate chains were extracted from the cell layer and conditioned medium of vascular endothelial cells, and their disaccharides were separated as in our previous experiment [29 (link)]. Briefly, vascular endothelial cells were treated with PMTAS (10 µM) for 48 h followed by proteoglycan extraction from the cell layer and conditioned medium under dissociative conditions in the presence of 8 M urea. The extract was concentrated on 0.3 mL DEAE-Sephacel minicolumns and precipitated with 3.5 volumes of 1.3% potassium acetate in 95% ethanol. The dried precipitate was digested overnight with proteinase K (800 µg/mL) in 0.1 M sodium acetate buffer (pH 7.2) at 60 °C. The glycosaminoglycan chains recovered with Microcon YM3 (3000 MW cut-off) ultrafiltration devices (Millipore, Billerica, MA, USA) were digested at 37 °C for 8 h with a mixture of heparinase II and heparinase III (0.03 U/mL each). The dried heparan sulfate samples were fluorotagged with 2-aminoacridone hydrochloride (0.1 M). The fluorotagged heparan sulfate hydrolase products were separated via electrophoresis, and the fluorescent images were displayed on a gel documentation system AE-6914 by ATTO (Tokyo, Japan). The bands of heparan sulfate unsaturated disaccharides were quantitatively analyzed using the NIH Image Analysis Software (ImageJ version 1.53k) using the bands of unsaturated GlcA-GalNAc(6S) as standards.

BCC/WWOX, BCC/WWOXsi, or BCC/WWOX scramble cells were plated in 1 ml culture medium without serum at 2 × 10⁵ cells per well in 24-well 18 mm culture dishes. The culture supernatants were collected 24 h later and centrifuged sequentially at 12,500 × g with Microcon YM-3 centrifugal filter devices (cutoff molecules smaller than 3000Da; Millipore) for 10 min to obtain a 10-fold concentrate culture supernatant.

Paraffin sections (5 μm) of lymph node tissue were labelled with anti-p41 Ii mAb as described [49 (link)]. Total tissue lysate was prepared from non-fixed tissue previously frozen in liquid nitrogen. Pieces of the latter, in 0.01 M phosphate buffer (pH 7.2), were sonicated using a Branson Digital Sonifier W-450 (Branson). The non-soluble fraction was pelleted by centrifugation. The supernatant containing soluble proteins was dialyzed in 0.01 M phosphate buffer using Microcon YM-3 (Millipore). Proteins were separated on SDS-PAGE and checked for the presence of p41 Ii.