Mouse anti p70s6k

RCC cells (5,000) were plated in 8-well chamber slides overnight and treated with HCQ (75 μM) or RAD001 (10 μM) for 2 days. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100 in PBS, and stained with following antibodies; rabbit anti-phospho S6 (Ser235/236, Alexa Fluor 488 conjugated) (Cell Signaling Technology), mouse anti-S6 (Cell signaling Technology), mouse anti-phospho-P70S6K (Thr389) (Millipore), and mouse anti-P70S6K (Santa Cruz Biotechnology). Alexa Fluor 647 conjugated anti-mouse IgG antibody (Life Technology) was used for secondary antibody. Nuclear DNA was visualized using a DAPI mounting solution (Vector Laboratories).

BMT (#B3023), TMZ (#T2577), propidium iodide (PI, #P4864), and MTT (#M2128) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat# 12430-054) and Penicillin/streptavidin (Cat# 15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from invitrogen (Carlsbad, CA). Anti-phospho-NKCC1(pThr206) antibody, anti-phospho-SPAK/OSR1 (pSer383 SPAK/pSer325 OSR1) antibody, and anti-total-SPAK/OSR1 (tSPAK/tOSR1) antibody were developed by Dr. Yang (Taiwan National University) and validated in previous studies (Moriguchi et al., 2005 (link); Yang et al., 2010 (link)). Monoclonal anti-total NKCC was from the Developmental Studies Hybridoma Bank (T4, Iowa City, IA). Antibody against α-tubulin (Cat #2125), rabbit anti-phospho AKT (Ser473; Cat# 9271), rabbit anti-AKT (Cat# 4691), rabbit anti-phospho ERK (Thr202/Tyr204; Cat# 4370), rabbit anti-ERK (Cat# 4695), and rabbit anti-phospho p70 S6k (T389; Cat# 9234) were from cell signaling (Beverly, MA). Mouse anti-p70 S6K (Cat# sc-8418) was purchased from Santa Cruz Biotechnology (Dallas, TX). BCA Protein Assay Kit (Cat #23227) was from Thermo Scientific (Rockford, IL). STS66 was synthesized by Töllner et al. (2014) (link) as described previously.