Affi gel blue

Affi-Gel Blue (cross-linked agarose beads with covalently coupled Cibacron Blue F3-GA) was purchased from Bio-Rad laboratories Inc. and mixed with the DSP peptide (2 mg/ml). The mixture was centrifuged and pellet used for implantation. The experimental procedures were approved by the animal research committee of UTHSCSA. Twenty-five adult mice (C57BL/6) aged 1.5 months were anaesthetized by sodium pentobarbital (Sigma-Aldrich, USA). The first molars on the maxilla were cleaned by sterilized instruments. Exposed pulped cavities were prepared by ¼ diamond cylindrical burs with sterile saline cooling. The exposed pulped cavity on the left first molars was filled onto Affi-Gel Blue gel as control and Affi-Gel Blue gel with the DSP peptide on the right first molars. The cavities over the implanted materials were filled with Vitremer Glass Ionomer (GI) Core Build-Up/Restorative (Sku# 3303PEDO, 3 M ESPE, Dental Products, St. Paul, NM, USA) and sealed by light curing. Sealant well done in each animal was observed carefully. The animals were euthanatized at 1, 3, and 5 weeks after surgery. Tissue specimen were fixed in 4% paraformaldehyde at 4 °C overnight and embedded in paraffin wax after demineralization with 10% EDTA. The paraffin sections (5 μm in thickness) were morphologically examined after staining with hematoxylin and eosin (HE) (Agilent Technologies, USA).

The human RPA plasmid was a gift from Dr. Marc Wold. The E. coli strain BL21 (DE3) was transformed with the plasmid p11d-tRPA, permitting the co-expression of RPA70, RPA32, and RPA14. RPA was then purified over Affi-Gel Blue, Hydroxyapatite (Biorad), and Q-Sepharose chromatography columns as described previously (Henricksen et al., 1994 (link)). The purified protein was eluted in phosphate buffer containing 300 mM KCl (pH 7.5). To obtain fluorescent human RPA, a DNA fragment encoding a variant of the enhanced GFP (eGFP) with a polyhistidine tag was inserted in frame at the 3′ end of the cDNA encoding the large subunit of RPA in the expression plasmid p11d-tRNA. eGFP-RPA purification was performed as previously described (van Mameren et al., 2009 (link)).

Samples of highly purified Fred were prepared at 4 °C using an LKB Biologic FPLC system deploying successive anion-exchange (Mono-Q, Pharmacia, Stockholm, Sweden), negative affinity (Reactive Blue-4-agarose, Pharmacia) and gel filtration (HiLoad 16/60 Superose 12, Pharmacia) columns in the three-stage protocol fully described by Willetts and Kelly [21 (link)]. A combined Frp1 plus Frp2 preparation was a by-product of the procedure, and this was resolved into separate highly purified Frp1 and Frp2 activities using the purification protocol deploying successive gel filtration (Sephadex G-25, Pharmacia) and affinity chromatography (Affi-Gel Blue, Bio-Rad, Hercules, CA, USA) steps developed and fully described by Halle and Meyer [27 (link)].

For FGF9 treatment, FGF9 (R&D systems, 0.1 mg/ml)-soaked beads (Affi-gel blue, Bio-Rad, ~ 50  μm) were injected into the interstitial regions of W/W^v mouse testes as previously reported^{13 (link),34 (link)}. The beads were labeled with DiI prior to the transplantation, which makes it distinguishable in the section. At 2 ~ 24 h after the bead treatment, testes were isolated for the subsequent histology and immunohistochemical analyses.
For RA treatment, wild-type male mice (ICR, 7–8 weeks old) were injected with beads (BioMag Amine) soaked in 40.0 mg/ml all-trans RA (Sigma-Aldrich) in 16% (vol/vol) dimethyl sulfoxide (DMSO) or DMSO (Fig. 5a). In this experiment, the beads were injected locally around the SV region (Fig. 5a) to examine the effects of locally elevated RA levels on the SV function. At 1 and 3 days after the treatment, testes were isolated for subsequent histology and immunohistochemical analyses.

RPA complex was expressed and purified as per the protocol from the M.S. Wold lab (27 (link),29 (link)). p11d-tRPA (123) plasmid (Supplementary Table S1) was transformed into E. coli BL21(DE3) strain. Cells were grown in the presence of Ampicillin antibiotics. Protein expression was induced at OD₆₀₀ of 0.7–0.8 by adding 0.3 mM of IPTG, and the culture was incubated at 37°C for 3 h. Protein purification was performed through Affi-gel blue (Bio-Rad; Cat. No. 153-7302), Hydroxyapatite (Bio-Rad; Cat. No. 130–0420), and Q-column (GE). 1.5 M NaSCN (Sigma; Cat. No. 251410), 80 mM Potassium phosphate, and 300 mM KCl buffer were used to elute the RPA from Affi-gel blue, Hydroxyapatite, and Q-column, respectively. Finally, dialysis of RPA was carried out in the NMR buffer containing 100 mM NaCl (20 mM Tris–HCl pH 7.4 at 25°C, 100 mM NaCl, 2 mM DTT, 0.02% NaN₃). SDS-PAGE analysis was performed to ascertain the purity, molecular weight and integrity of the trimeric complex RPA (Figure S3B). The concentration was calculated by UV absorbance at 280 nm and using the calculated molar extinction coefficients of RPA (ϵ = 87210 M⁻¹ cm⁻¹ for trimeric complex) determined through EXPASY webserver (28 (link)).

Recombinant human aldose reductase was purified from bacterial cultures. Enzyme from expression cultures was extracted and purified essentially as described previously (12 (link)) with the exception that affinity chromatography over AffiGel Blue (Bio-Rad) was used as a final purification step.