Anti transferrin

Liver samples from Hnf4a^f/f and Hnf4a^ΔH mice were homogenized in lysis buffer (7 m urea, 2 m thiourea, 1% Triton X-100) and allowed to sit on ice for 30 min. The homogenate was centrifuged at 12,000 × g for 30 min at 4 °C, and the supernatants were used as whole cell lysates. The whole cell lysates and serum protein (40 μg), determined by Quick Start^TM Bradford Dye Reagent (Bio-Rad), were diluted with Laemmli sample buffer, incubated at 65 °C for 15 min, and fractionated by 10% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue R-250 or transferred onto a PVDF membrane (GE Healthcare, Tokyo, Japan). The membrane was incubated for 1 h with PBS containing 0.1% Tween 20 and 5% skim milk and then incubated for 1 h with anti-transferrin (Santa Cruz Biotechnology, Dallas, TX), anti-transferrin receptor 2 (Abcam, Tokyo, Japan), and anti-γ-tubulin (TUBG) (Sigma) antibodies. After washing, the membrane was incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), and the reaction product was visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Expression of TF and TFR2 proteins was quantified by densitometric analysis using ImageJ software and the expression in Hnf4a^ΔH was presented as expression differences relative to the Hnf4a^ff/f mice.

GSS was purchased from the HANKOOK SHINYAK Corporation (Chungcheongnam-do, South Korea). Primary antibodies used for Western blotting were as follows: anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1 : 1000; Wako, Japan), anti-glial fibrillary acidic protein (GFAP; 1 : 3000; Millipore, MA, USA), anti-survival motor neuron (SMN; 1 : 1000; Santa Cruz Biotechnology, CA, USA), anti-TLR4 (1 : 1000; Santa Cruz Biotechnology), anti-CD14 (1 : 1000; BD Pharmingen, CA, USA), anti-COX2 (1 : 1000; Abcam, MA, USA), anti-transferrin (1 : 1000; Santa Cruz Biotechnology), anti-HO1 (1 : 1000; Abcam), anti-Bcl2 associated X (Bax, 1 : 1000; Santa Cruz Biotechnology), anti-phospho 5′-adenosine monophosphate-activated protein kinase (pAMPK; 1 : 1000; Cell Signaling, MA, USA), anti-AMPK (1 : 1000; Cell Signaling), and anti-phospho mammalian target of rapamycin (mTOR; 1 : 1000; Cell Signaling). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 1000; Santa Cruz Biotechnology) was used to control for protein loading. Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

Total protein concentration of oligomers from PTS from the sera of either controls or patients included in the study was determined by BCA method, and the concentration of the samples was normalized. WB assays were done as previously described [9 (link)] briefly the PTS were loaded (30 μg) and run in 15% bis-tris SDS-PAGE gels. The membranes were probed overnight at 4°C with the novel anti-hIAPP oligomer antibody (MEX1) (1:800) or the purified anti-amyloid oligomer antibody ab126892-A11 (Abcam, cat AB-126892 1:1,000) or anti-amylin antibody (Santa Cruz Biotechnology, cat. SC-57026, 1:200) diluted in phosphate-buffered saline with Tween-20 (PBS-T). To control the charge, we performed immunoblotting with anti-transferrin (Santa Cruz Biotechnology, cat. sc-30159; 1:500). The membrane activity was detected by substrate chemiluminescence (Immobilion Chemiluminescence HRP Substrate 1:1) and revealed by a Li-COR C-DiGit system. The intensity of the protein bands was quantified by Image Studio Lite via scanning densitometry. The data were managed with MS Excel, while the statistics and graphs were obtained with the SAS JMP 9 statistical software package.