Anti pak4

Cells were starved and pretreated with U0126 (5 μM), SB203580 (5 μM), H89 (10 μM), SP600125 (5 μM) or wortmannin (0.5 μM) for 1 h and then stimulated with 10 nM TSH for 60 min. Cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA and 1% Triton-X 100) with a protease inhibitor cocktail for 30 min at 4°C. Lysates were quantified using a BCA protein assay kit (Pierce) according to the manufacturer's instructions. Proteins in the lysate were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). After blocking with 5% skimmed milk, the membranes were incubated with anti-PAK4 (1:1000), anti-p-PAK4 (1:1000), anti-PKA Cα(1:1000) (Cell Signaling Technology, Beverly, MA, USA), anti-TSHR (1:1000) (Santa Cruz), cAMP (1:1000) (Sigma) and anti-GAPDH (1:2000) (GenScript Corporation, Nanjing, China) antibodies. U0126, SB203580, H89, SP600125, wortmannin, and forskolin were purchased from BioTime Technology.

Unless otherwise stated, general lab reagents were purchased from Sigma Aldrich and were of the highest quality available. The following primary antibodies were used for immunoblotting at 1/1000 dilution in 5% (w/w) BSA overnight at 4°C: anti-RelA (sc-372, Santa Cruz), anti-HA (Merck, HA-7), anti phospho-histone H2AX (#2577, Cell Signalling), anti-PAK4 (#3242, Cell Signaling Technologies), anti-Karyopherin-β1 H-7 (sc-137016, Santa Cruz). Secondary anti-rabbit IgG (7074S, Cell Signalling Technology) was used at 1/5000 dilution following membrane incubation with VeriBlot (1/400 dilution, ab131366 Abcam).

Immunohistochemical staining was performed using a standard streptavidin-biotin-peroxidase complex method (EnVision™ Detection System; Dako, Copenhagen, Denmark). Tissue blocks were cut into 5-mm sections, deparaffinized with xylene, and rehydrated in a graded ethanol series. The sections were stained with anti-PAK4 (1:50; Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C.
The degree of immunostaining was reviewed and scored semiquantitatively by two independent observers. The staining index was calculated as the product of the proportion of positively stained tumor cells and the staining intensity. The former was scored as follows: 0 (0 % positive tumor cells), 1 (<10 %), 2 (10–35 %), 3 (35–70 %), and 4 (>70 %). Staining intensity was graded as follows: 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). The staining index was scored at 0–12. The cutoff values for high and low PAK4 expression were ascertained by measuring heterogeneity using log-rank testing with respect to overall survival. A staining index score of ≥6 indicated high PAK4 expression; a staining index score of <6 indicated low PAK4 expression.

The 24ST1NLESG cells were transfected by electroporation using the Neon Transfection System (Life Technologies, Carlsbad, CA, USA). Electroporation parameters were 1350 Pulse Voltage (V), 10 Pulse Width (ms), 3 Pulse Number, 2 × 10⁷ cell/mL. Final concentrations were 1 × 10⁷ cell/mL, 1 µM siRNA (siPAK1, Dharmacon, cat # L-003521-00-0020; siPAK2, Dharmacon, cat # L-003597-00-0020; siPAK4, Dharmacon, cat # L-003615-00-0020; siMAPK1, Thermofisher cat # 4390824; siPKA, Thermofisher cat # 4390825; Non-targeting Control, Dharmacon, cat # D-001810-10-20). The efficiency of siRNA knockdown was assessed by Western blot analysis. Anti-PAK1 (cat # 2602S), anti-PAK2 (cat #2608S), anti-PAK4 (cat # 62690), anti-PKA (cat # 4782), anti-MAPK (cat # 9108), and anti-GAPDH (cat # 97166S) antibodies were purchased from Cell Signaling. The efficiency of siRNA knockdown was quantified using ImageJ.

The prepared cells and tissues were lysed for 30 min on ice in RIPA lysis buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% deoxycholate, supplemented with the protease inhibitor PMSF. After being centrifuged at 14,000×g for 30 min at 4 °C the supernatants were collected. SDS-polyacrylamide gelelectrophoresis and western blot were performed according to standard protocols. Human and mouse monoclonal antibodies of anti-PAK4, anti-p-MEK, anti-p-ERK and anti-β-actin (Cell Signaling Technology) were all diluted at 1:1000. Secondary antibodies were all diluted at 1:4000.

Cells were cultured on cover glasses, fixed using paraformaldehyde, and permeabilized with 0.1 % Triton X-100 in TBS. The cover glasses were incubated with the primary antibodies (anti-PAK4, Cell Signaling Technology; anti-LIMK1, Cell Signaling Technology) at 1:50 dilutions. PAK4 was detected with an anti-goat secondary antibody conjugated to Alexa Fluor 488 (Invitrogen Life Technologies). LIMK1 was detected with an anti-rabbit secondary antibody conjugated to Alexa Fluor 555 (Invitrogen Life Technologies). The fluorescent staining was visualized using a 63× NA 1.3 oil objective on a confocal microscope (LSM 510 Meta; Carl Zeiss, Inc.). The co-localization was analyzed using Pearson’s correlation coefficient (full co-localization = 1.0) by the Image Pro Plus software.

The standard streptavidin-biotin-peroxidase complex method (EnVision™ Detection System; Denmark) was used to perform immunohistochemical staining [60 (link)–63 (link)]. NSCLC tissues were cut into 4-mm sized sections, xylene was utilized to deparaffinize them, and graded ethanol series was used for rehydration. To stain them, they were treated with anti-PAK4 (1:500; Cell Signaling Technology) or GRP78 (1:500; Abcam) and subjected to incubation at 4 °C overnight. Two skilled observers independently graded the degree of immunostaining. The intensity and frequency of staining were estimated as previously described [20 (link)]. The staining index scores ranged from 0 to 12.