Non fat powdered milk

Proteins were extracted from miR-204-, miR-211-, or miR-negative control-transfected MCF-7 cells using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) with a protease inhibitor cocktail (Thermo Fisher Scientific). Diluted protein samples were separated on 4–12% gradient SDS-PAGE gels (Koma Biotech, Korea) and transferred to polyvinylidene difluoride membranes (Whatman, UK). Then, the membranes were blocked in 5% non-fat powdered milk (Bio Basic, Canada) for 1 h and incubated for 1 h with anti-MX1 (1:500, Santa Cruz, Dallas, Texas, USA) and anti-TXNIP (1:500, Novus, St. Charles, MO, USA) antibodies. The membranes were then rinsed in 0.01% Tween-20 TBS five times for 5 min each and incubated with horseradish peroxidase conjugated secondary donkey anti-rabbit antibody (1:5000, Amersham, UK) for 1 h. The bands were detected by an EzWay ECL Western Blot Substrate Kit (Koma Biotech) and Image Lab software (Bio-Rad, Hercules, CA, USA). As an endogenous reference, rabbit polyclonal anti-β-actin antibody (1:500) (Novus) was used.

A nitrocellulose membrane was placed in the Bio-Dot Apparatus (Bio-Rad, Mississauga, ON, Canada) and was rehydrated by injecting 100 μL of a tris-buffered solution (TBS) into each well. Total protein extract (5 µg or 10 µg) was loaded in the wells, and then each well was rinsed twice with 200 µL of TBS. The nitrocellulose membrane was removed from the device and rinsed in TBS with 0.1% Tween-10 solution (TBS-T). The antigenic sites were blocked for 1 h in TBS-T with a 5% powdered milk solution (Non-Fat Powdered Milk, Bio Basic, Markham, ON, Canada). The membranes were incubated for 1 h with the primary antibodies and for an additional hour with the secondary antibodies (Table S1). The proteins of interest were detected using an ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and the Fusion F × 7 imager (MBI Lab Equipment, Kirkland, QC, Canada). Quantification of the dot blots was performed through densitometry using ImageJ (Wayne Rasband, National Institute of Health, USA).