Trib1

Proteins were mixed with 5 × Laemmli buffer and incubated at 100 °C for 10 minutes. Samples were immediately transferred in the ice afterwards. All samples and prestained protein ladder (10-250 kDa, Thermo ScientificTM) were loaded into the columns of NuPAGETM 4-12% Bis-Tris Gel (Invitrogen) placed in the Invitrogen tank containing 1× NuPAGE MOPS SDS running buffer (Novex). The gel was run at 100v for 75 minutes and transferred to a PVDF (Polyvinylidene difluoride) membrane (Millipore) using NuPAGE transfer buffer (Novex) with methanol and antioxidant (Invitrogen) at 35v for 60 minutes. The membrane was blocked with 5% milk-TBST at RT for 1 hour and incubated overnight with TRIB1 (Millipore), and HSP90 (Abcam) diluted in 5% milk-TBST (1:1000 and 1:5000 respectively) at 4 °C. The membrane was then washed with 0.1 v/v TBST for 5 minutes 3 times and incubated with Polyclonal Goat anti-Rabbit Immunoglobulin/HRP, and Polyclonal Rabbit anti-Rat Immunoglobulin/HRP (Dako) diluted in 5% milk-TBST (1:2500 and 1:5000 respectively) at RT for 1 hour. The membrane was then washed with TBST 3 times for 5 minutes, incubated with ECL, and imaged with Bio-Rad imager.

The frozen tumor sections were adjusted to RT and then flooded with ice-cold acetone for 10 minutes to fix the tissue. The slides were then air-dried and rehydrated in 0.5% Tween-PBS (PBST) for 3 minutes. The non-specific binding of the secondary antibody was blocked with serum-free protein block (Dako X0909) at RT for 30 minutes and incubated with the following antibodies at 1:25 dilution: F4/80 Alexa Fluor 488 (Bio-rad); 1:50 dilutions: NOS2 (Abcam), CD3 APC (Tonbo Bioscience), CA9 (Abcam), CD68 (Abcam) and TRIB1 (Millipore); 1:100 dilutions: CD31 Alexa Fluor 674 (Biolegend), MR (Abcam), CD4 Alexa Fluor 488 (Biolegend), CD8 PE (Biolegend), IL-15 (Abcam) for 1 hour at RT. The samples were washed twice with PBST for 5 minutes and then incubated with secondary antibody Goat anti-Rabbit IgG (H&L) Dylight 550 (ImmunoReagents), both at 1:50 dilution, Alexa Fluor 488 or 594 goat anti-mouse-IgG or anti-rabbit-IgG secondary antibodies (Invitrogen) at 1:1000 for human TNBC sample, for 1 hour at RT. Slides were washed three times with PBST for 5 minutes and mounted with Antifade mounting medium with DAPI (Life Technology). Slides were kept in the dark overnight at RT and imaged immediately or stored at 4 °C. Random areas of 4-5 images were captured using a Leica AF6000 microscope, or Nikon A1 confocal microscope and cells were manually quantified with ImageJ (NIH).