Txnip d5f3e

Tumors and cell lysates were prepared as previously described in Ref. [19 (link)]. Total protein extracts (40 μg) were separated under reducing conditions on 5–15% polyacrylamide gels (depending on the molecular weight) and transferred onto polyvinylidene difluoride membranes (NEN, Boston, MA, USA). Membranes were saturated for 1 h with casein (1%, w/v) in PBS-Tween-20 (0.1%, v/v). Antigenic bands were detected by exposing the membranes to human primary antibodies targeting the following proteins: SCD1 [M38], SCD1 [ab52356, Abcam], TxNIP [D5F3E], Cell Signaling (Beverly, MA, USA), FABP4 [ab13979], GPX4 [ab231174] Abcam (Cambridge, England), SOD1 [8B10], Catalase [PA5-23246] Thermofisher (Rockford, IL, USA), GPX1 [A0873] and serotransferrin [A1448] (Abclonal, Woburn, MA, USA). After washes, membranes were incubated with a secondary horseradish peroxydase (HRP)-conjugated goat anti-rabbit antibody (1:2000, DakoCytomation) or sheep anti-mouse antibody (1:1000, DakoCytomation). Immunocomplexes were visualized by chemiluminescence reaction on a luminescent image analyzer (LAS-4000; Fujifilm, Wavre, Belgium). For loading control, membranes were stripped and re-incubated with Hsc70 [B-6, sc-7298] antibody (Santa Cruz, CA, USA), or GAPDH [MAB37A] and actin [A2066] antibody (Sigma Aldrich, Saint-Louis, USA).